Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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PYRIMIDO[4,5-NINDOLE DERIVATIVES AND USE THEREOF IN THE EXPANSION OF
HEMATOPOIETIC STEM CELLS
FIELD OF THE INVENTION
[0002] The invention relates to pyrimido[4,5-b]indole derivatives. Also, the
invention relates to use of
pyrimido[4,5-b]indole derivatives for expanding hematopoietic stem cells.
Moreover, the invention
relates to medical treatments of diseases involving hematopoietic stem cells.
BACKGROUND OF THE INVENTION
[0003] The main sources of hematopoietic stem cells (HSCs) are the bone marrow
and the umbilical
cord blood (UCB). HSCs are used in the transplantation setting (autologous or
allogeneic) which
constitutes one of the most effective treatment strategies for achieving cures
in patients with
hematologic malignancies, bone marrow failure conditions, a variety of
congenital diseases of global
concern (e.g. sickle cell anemia and thalassemia) and autoimmune diseases such
as lupus. However,
this opportunity for life-saving or life-improving treatment is not available
to many thousands of people
worldwide due to an inability to amplify these cells ex vivo sufficiently to
make the procedure safe and
successful. More particularly, for every 3 patients, one will forego the
opportunity for transplant
because no human leucocyte antigen (HLA) identical donor can be found. Another
proportion of
patients will not have access to transplantation simply because too few HSCs
are available in the graft
(i.e. cord blood or autologous) for successful transplant. The safety and
efficacy of marrow transplant is
directly dependent on the number of HSCs and progenitor cells available for
engrafting. The more that
can be infused, the more rapidly is hematologic function restored, and the
shorter is the window of risk
for infection due to lack of granulocytes or of bleeding due to lack of
platelets. The challenge in
providing sufficient HSCs is further escalated where non-myeloablative
conditioning is preferred such
as in the context of gene therapy for major inherited blood disorders (the
major genetic cause of
morbidity and mortality worldwide).
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[0004] In adults, HSCs mainly reside in the bone marrow and must be mobilized
to enter the
circulation prior to being collected by apheresis, either for autologous or
allogeneic hematopoietic stem
cell transplantation (HSCT). The collection of an adequate number of CD34+
cells, a surrogate marker
of (HSCs), is paramount because the dose of CD34+ cells influences the success
and rate of
hematopoietic recovery. Several
reports suggest that a higher infused CD34+ cell dose is
independently predictive of improved survival.
[0005] The two most commonly used mobilizing regimens are granulocyte-colony
stimulating factor
(G-CSF) and G-CSF plus chemotherapy. Plerixafor, a CXCR4 antagonist approved
by the United
States Food and Drug Administration (FDA) in 2008 and in 2011 by Health
Canada, enhances
mobilization of HSCs when administered with G-CSF. However, Plerixafor is
contraindicated in patients
with leukemia because of mobilization of leukemic cells. Inability to obtain
sufficient numbers of CD34+
cells/kg with currently used mobilization regimens is estimated to affect up
to 15% of patients (varies
between diseases). Use of autologous HSCT in hematological malignancies is
often limited by the fact
that both normal and cancer stem cells are present in the bone marrow and
thus, likely to be mobilized.
[0006] Allogeneic HSCT with BM or mPBSC is another transplantation
alternative. However, about
one third to one fourth of the patients who are eligible for this type of
transplant cannot find a suitable
donor. For those who get transplanted, there is a high frequency of transplant
related mortality due to
graft-versus-host disease, relapse or graft rejection; and a risk of
immunodeficiency for prolonged
periods of time. Alternatively, umbilical cord blood has been shown as a valid
option in allogeneic
HSCT. However, a single CB unit typically provides insufficient HSCs for an
adult patient for a rapid
and efficient hematopoietic recovery.
[0007] In vitro conditions that support cytokine-mediated short-term
maintenance or even modest
increase in murine or human HSC numbers measured by mouse reconstitution
assays are generally
accompanied by much more robust increase in later types of progenitor cell
populations. More marked
increases in murine and human HSCs have more recently been described in
cultures containing other
factors such as fibroblast growth factor (FGF), insulin-like growth factor
binding proteins, angiopoietin-
like growth factors and pleiotrophin. However, these latter reports are thus
far solitary and await
independent confirmation. Short-term increases in HSCs obtained with standard
cytokines in vitro are
also inevitably followed by eventual HSC depletion.
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[0008] Alternative strategies for human HSC expansion have involved their
culture with stromal
elements or soluble morphogenic ligands (e.g. stimulating the Notch, Wnt and
Hedgehog pathways),
targeted manipulation of specific intracellular signaling pathways (PGE2, ROS,
p38 and MAPK
inhibitors) or manipulation of specific transcription factors (e.g. Hox, Hlf).
Other preclinical approaches
for ex vivo expansion of HSCs include incubation with: i) StemRegenin1 (SRI),
an aryl hydrocarbon
receptor antagonist (Boitano, AE et al. "Aryl hydrocarbon receptor antagonists
promote the expansion
of human hematopoietic stem cells" Science 329: 1345-1348. 2010); ii)
Garcinol, a histone
acetyltransferase inhibitor (Nishino, T et al. "Ex vivo expansion of human
hematopoietic stem cells by
Garcinol, a potent inhibitor of histone acetyltransferase" PLoS ONE 6(9):
e24298. 2011); and iii) NR-
101, a non peptidyl small molecule c-MPL agonist (Nishino et al. "Ex vivo
expansion of human
hematopoietic stem cells by a small-molecule agonist of c-MPL" Exp. Hem. 2009;
37:1364-1377).
Characterization of SRI provided a proof of principle that low molecular
weight ([MW) compounds
have the ability to promote HSC expansion.
[0009] Clinical studies have stressed the requirement not only for permanence
of the administered
transplants, but also the importance of minimizing the time to appearance of
useful granulocyte levels
post-transplant which, in turn, depends on the number of short term
repopulating cells infused.
Transplantation of marrow or cord blood cells expanded in culture with
cytokines has not so far
demonstrated clinically useful acceleration of hematopoietic recovery compared
to untreated cells.
Early results of trials with cells expanded using immobilized Notch ligands
have been the first to show
potential clinical utility for any (even modest) progenitor cell expansion
strategy (Delaney et al. "Notch-
mediated expansion of human cord blood progenitor cells capable of rapid
myeloid reconstitution" Nat.
Med. 16(2): 232-236. 2010). This approach is however limited by the need to
use an immobilized
Delta-1 fusion protein during the ex vivo expansion step and by the lack of
documented effect on stem
cells (impact appears limited to more differentiated progenitors). Other
approaches in clinical trial
include: i) StemEx, a combination of UCB cells cultured with the copper
chelator
tetraethylenepentamine (TEPA) and cytokines, co-infused with non-treated UCB
cells; phase I results
show that time to neutrophil or platelet engraftnnent was not improved
compared to previous reports (de
Lima M et al. "Transplantation of ex vivo expanded cord blood cells using the
copper chelator
tetraethylenepentamine: a phase I/II clinical trial" Bone Marrow Transplant.
2008; 41(9): 771-778); and
16-16 dimethyl prostaglandin E2 (PGE2), used for improving homing of UCBT in a
phase I trial.
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[0010] There is thus a need for novel strategies for increasing the expansion
of hematopoietic stem
and progenitor cells. Certain pyrimido[4,5-b]indole derivatives are known in
the art that are used in that
regard; they are disclosed for example in: WO 2003/037898; WO 2004/058764; WO
1998/042708; WO
1997/002266; WO 2000/066585; WO 1993/020078; WO 2006/116733; WO 2008/055233;
WO
2010/006032; WO 1995/019970; WO 2005/037825; and WO 2009/004329. However,
these
documents do not disclose the pyrimido[4,5-b]indole derivatives according to
the invention or their use
in the expansion of hematopoietic stem and progenitor cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] Figure 1: Compoundl does not act through aryl hydrocarbon (AhR)
pathway. Mobilized
peripheral blood CD34(+) cells were cultured for 12 hours with DMSO, SR1 [AhR
antagonist 1000nM],
and Compound1 [500nM], cells were harvested and real-time quantitative RT-PCR
for AhR-responsive
genes (CYP1B1 and AhRR) was performed. Compound 1 unlike SRI, does not
suppress AhR-
downstream target genes suggesting that its function is independent on AhR
pathway.
[0012] Figure 2: Effect of connpound1 is reversible. Mobilized peripheral
blood CD34(+) cells cultured
for 7 days with compound1 (shown in green) or vehicle (DMSO, shown in blue)
were washed and re-
plated in fresh media with or without Compound addition (solid vs dashed line
respectively). Cells were
cultured for 8 additional days and the percentage of CD34+CD45RA- was
monitored. A rapid decrease
in CD34+CD45RA- percentage was observed when compound1 was washed out
indicating that its
effect is reversible.
[0013] Figure 3: Flt3,SCF, and TPO are required for compoundl mediated stem
cell expansion.
Mobilized peripheral blood CD34(+) cells were cultured for 7days in the
presence or absence of the
growth factors (F1t3+SCF+TP0). CD34+CD45RA- cell count was lower in the
absence of any of the
growth factors suggesting their requirement for the observed effect.
[0014] Figure 4: A) Compound 1 reduces differentiation of CD34+ mobilized
peripheral blood cells. As
determined by FACS analysis, compound1 was able to expand CD34+ cell
population compared to
DMSO treated control cells. SRI synergizes with compound1 to keep the expanded
culture cell CD34+
suggesting that the inhibition of differentiation is even more prominent with
the combination. B)
Compound 1 reduces differentiation of CD34+ cord blood cells. C) Compound 40
reduces differentiation
of CD34+ cord blood cells. D) Compound 40 exhibits a dose-dependent effect on
suppression of
0D34+ cell differentiation.
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[0015] Figure 5: A) Compound 1 expands mobilized peripheral blood-derived
CD34+ and
CD34+CD45RA- cell populations ex vivo. Although the total cell count was the
same in DMSO and
compound 1 treated cells, CD34+ population was more than twice that of DMSO.
Similarly,
CD34+CD45RA- population was more than three times in the compound 1 treated
group vs. DMSO.
This observation was enhanced with the co-treatment with SRI. B) Compound 1
enhances expansion
of the cord blood-derived CD34+ and CD34+CD45RA- cells during 7-day
incubation. C) Compound 1
exerts the positive effect on the in vitro expansion of the cord blood-derived
CD34+ and
CD34+CD45RA- cells during 12-day incubation.
[0016] Figure 6: CD34+ mPB cells were cultured for ten days in the presence of
the vehicle (DMSO),
SRI, compound 1, and a combination of compound1 and SR1. The outcome of 50,000
and 500,000
treated cells were transplanted in NSG mice and the bone marrow analysis was
performed after 13
week post-transplantation to evaluate the human hematopoietic engraftment. At
any give cell dose,
0D34+ mPB cells treated with Compound 1 engrafted better than that of DMSO.
Interestingly, the
percentage of the human CD45+ cells in the BM was highest in NSG mice which
received cells treated
with the combination (compound 1+SR1) compared to the individual compounds.
[0017] Figure 7: A) Compound 1 expanded cells are capable of reconstituting
human hematopoietic
cells in NSG mice. The outcome of 5,000 CD34+ CB Cells treated with vehicle
(DMSO), SRI,
compound1 and combination were transplanted in immunocompromised NSG mice.
After 8- weeks
post transplantation, the bone marrow analysis showed a human CD45+
engraftment in all treatment
groups except DMSO. The cells treated with the combination (compound1+SR1)
showed the highest
engraftment levels. B) During 12-day in vitro incubation Compound 40 prevents
the loss of human
hennopoietic cells with ability to engraft NSG mouse bone marrow.
[0018] Figure 8: Expansion of primitive cell phenotypes (CD344, CD34+CD90+,
and CD34+CD45RA+)
in bioreactor using the fed-batch approach as measured by flow cytometry. FB
control =fed-batch
without Compound 40, Cpd 40= fed-batch with Ccompound 40.
SUMMARY OF THE INVENTION
[0019] The inventors have discovered certain pyrimido[4,5-b]indole
derivatives. These compounds
are useful to expand hematopoietic stem cell populations, particularly, human
hematopoietic stem cell
populations. The compounds are also useful in the medical treatment of
diseases that involve
hematopoietic stem cells.
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[0020] According to an aspect, the invention provides for compounds of the
following general formulas
I, 11,111, IV, V and VI:
I II
I N N
,----R2
¨N
N
I N N
---N ¨N
R4R3N ( Li-XI R4R3N (
III Iv
\ N., N
I N N
¨N ¨N
X.L
,2cNR1 .ANR1
2 2
V VI
[0021] The substituents in the above general formulas 1, II, Ill, IV, V and
VI, namely, Z, W, L, Li, X, Xi,
R1, R2, R3, R4 and m are as defined herein below.
[0022] According to an aspect, the invention provides for pharmaceutical
compositions comprising a
compound of general formula I, II, III, IV, V or VI.
[0023] According to an aspect, the invention provides for use of a compound of
general formula 1, 11,
III, IV, V or VI to expand hematopoietic stem cells. In embodiments of the
invention, the hematopoietic
stem cells are human cells.
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[0024] According to an aspect, the invention provides for a method of
increasing hematopoietic stem
cells or progenitor cells, the method comprising culturing a starting cell
population in the presence of a
compound of general formula I, II, Ill, IV, V or VI. In embodiments of the
invention, the starting cell
population is in vivo, in vitro or ex vivo. Also, in embodiments of the
invention, the starting cell
population comprises 0D34+ cells harvested from mobilized peripheral blood
(mPB), bone marrow
(BM) or umbilical cord blood (UCB). Moreover, in embodiments of the method
according to the
invention, optionally, culture of the starting cell population in the presence
of a compound of general
formula I, II, Ill, IV, V or VI in performed together with at least one cell
expanding factor which is a
biologic or another small molecule.
[0025] According to an aspect, the invention provides for a cell population
expanded according to the
method of the invention, more specifically, a cell population expanded using a
compound according to
the invention. In embodiments, the invention provides for hematopoietic stems
cells expanded
according to the method of the invention, more specifically, hematopoietic
stems cells expanded using
a compound according to the invention.
[0026] According to an aspect, the invention provides for a method of treating
a hematopoietic
disorder! malignancy, an autoimmune disease and/or an inherited
immunodeficient disease in a subject,
the method comprising administering to the subject in need of such treatment
hematopoietic stem cells
expanded using a compound of general formula I, II, Ill, IV, V or VI, or a
compound of general formula I,
II, Ill, IV, V or VI.
[0027] In embodiments of the invention, the hematopoietic disorder/malignancy,
the autoimmune
disease and/or the inherited immunodeficient disease comprise bone marrow
failure conditions, a
variety of congenital diseases of global concern (e.g. sickle cell anemia and
thalassemia), lupus, acute
myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia,
chronic lymphocytic
leukemia, myeloproliferative disorders, myelodysplastic syndromes, multiple
myeloma, non-Hodgkin's
lymphoma, Hodgkin's disease, aplastic anemia, pure red cell aplasia,
hemoglobinuria, Fanconi anemia,
thalassemia, sickle cell anemia, Wiskott-Aldrich syndrome, inborn errors of
metabolism (such as
Gaucher disease among others).
[0028] According to an aspect, the invention provides for a kit for use in
increasing stem or progenitor
cells or in expanding hematopoietic stem cells, the kit comprising a compound
of general formula I, II,
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III, IV, V or VI, and instructions for use. In embodiments of the invention,
the kit comprises at least one
cell expanding factor which is a biologic or another small molecule.
DETAILED DESCRIPTION OF THE INVENTION
[0029] The inventors have discovered certain pyrimido[4,5-b]indole
derivatives. These compounds
are useful to expand hematopoietic stem cell populations, particularly, human
hematopoietic stem cell
populations. The compounds are also useful in the medical treatment of
diseases that involve
hematopoietic stem cells.
[0030] The compounds according the invention have the general Formula I, II,
III, IV, V or IV shown
below. Salts or prodrugs of such compounds are also within the scope of the
compounds according to
the invention.
N
I N N
I II
--N --N
N
I N N
-
--N ¨N
R4R3N __________________________ R4R:Xi (
III Iv
N
I N N
)--R2
¨N
X,L X,L
NR1 cNR1
2 2
V VI
[0031] In formulas I, II, III, IV, V and VI, the substituents are defined as
outlined below.
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[0032] Z is: 1) -P(0) (OR1) (OR1), 2)-C(0)0R1, 3) -C(0)NHR1, 4) -C(0)N(R1)R1,
5) -C(0)R1, 6) -CN, 7)
-SRI, 8) -S(0)2NH2, 9) -S(0)2NHR1, 10) -S(0)2N(R1)R1, 11) -S(0)R1, 12) -
S(0)2R1, 13) -L, 14) -benzyl
optionally substituted with 1, 2 or 3 RA or R1 substituents, 15) -L-heteroaryl
optionally substituted with
one or more RA or R1 substituents attached on either or both the L and the
heteroaryl groups, 16) -L-
heterocyclyl optionally substituted with one or more RA or R1 substituents
attached on either one or both
the L and the heterocyclyl groups, 17) -L-aryl optionally substituted with one
or more RA or R1
substituents attached on either or both the L and the heteroaryl groups, 18) -
heteroaryl optionally
substituted with one or more RA or R1 substituents, or 19) -aryl optionally
substituted with one or more
RA or R1 substituents. In this list, each substituent is optionally attached
to the L group if it is not
already present; and, when (R1) and R1 are attached to a nitrogen atom,
optionally they join together
with the nitrogen atom to form a 3 to 7-membered ring which optionally
includes one or more other
heteroatom selected from N, 0 and S, optionally the ring is substituted with
one or more R1 or RA.
[0033] W is H, a halogen or a group that is attached to the pyrimido indole
core of the molecule
through an atom which is N, 0, S, or C. Optionally, W comprises at least one
moiety which is
saturated, unsaturated, linear, branched and/or cyclic alkyl and/or
heteroalkyl having 1 to 20 carbon
atoms. Also, optionally, the moiety comprises at least one other hetero atom
which is N, 0 or S. As will
be understood by a skilled person, W in the chemical structure of the
compounds according to the
invention can belong to various categories of chemical groups commonly used in
the art.
[0034] More specifically, W is: 1) -H, 2) -halogen, 3) -0R1, 4) -L-OH, 5) -
LORI, 6) -SR1, 7) -CN, 8) -
P(0)(0R1)(0R1), 9) -NHR1, 10) -N(R1)R1, 11) -L-NH2, 12) -L-NHR1, 13) -L-
N(R1)R1, 14) -L-SR1, 15 -L-
S(0)R1, 16) -L-S(0)2R1, 17) -L-P(0)(0P1)(0P1), 18) -C(0)0R1, 19) -C(0)NH2, 20)
-C(0)NHR1, 21) -
C(0)N(Ri)R1, 22) -NHC(0)R1, 23) -NR1C(0)R1, 24) -NHC(0)0R1, 25) -NR1C(0)0R1,
26) -0C(0)NH2,
27) -0C(0)NHR1, 28) -0C(0)N(R1)R1, 29) -0C(0)R1, 30) -C(0)R1, 31) -NHC(0)NH2,
32) -
NHC(0)NHR1, 33) -NHC(0)N(R1)R1, 34) -NR1C(0)NH2, 35) -NR1C(0)NHR1, 36) -
NR1C(0)N(R1)R1, 37)
-NHS(0)2R1, 38) -NR1S(0)2R1, 39) -S(0)2NH2, 40) -S(0)2NHR1, 41) -S(0)2N(R1)R1,
42) -S(0)R1, 43) -
S(0)2R1, 44) -0S(0)2R1, 45) -S(0)20R1, 46) -benzyl optionally substituted with
1, 2 or 3 RA or R1
substituents, 47) -L-heteroaryl optionally substituted with one or more RA or
RI substituents attached on
either or both the L and the heteroaryl groups, 48) -L-heterocyclyl optionally
substituted with one or
more RA or R1 substituents attached on either or both the L and the
heterocyclyl goups, 49) -L-aryl
optionally substituted with one or more RA or R1 substituents attached on
either or both the L and aryl
groups, 50) -L-NR1(R1), 51) -L-)2 NR1, 52) -L-(N(R1)-1_)7 - N(R1)R1, 53) -L-
(N(R1)-L)8 - heteroaryl
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optionally substituted with one or more RA or R1 substituents attached on
either or both the L and
heteroaryl groups, 54) -L-(N(R1)-L)0 ¨ heterocyclyl optionally substituted
with one or more RA or R1
substituents attached on either or both the L and heterocyclyl groups, 55) -L-
(N(R1)-L)0 ¨ aryl optionally
substituted with one or more RA or R1 substituents attached on either or both
the L and aryl groups, 56)
-0-L-N(R1)R1, 57) -0-L- heteroaryl optionally substituted with one or more RA
or R1 substituents
attached on either or both the L and heteroaryl groups, 58) -0-L- heterocyclyl
optionally substituted with
one or more RA or R1 substituents attached on either or both the L and
heterocyclyl groups, 59) -0-L-
aryl optionally substituted with one or more RA or R1 substituents attached on
either or both the L and
aryl groups, 60) -0-L)2-NR1, 61) -0-L-(N(R1)-L)8 ¨ N(R1)R1, 62) -0-L-(N(R1)-
L)0 ¨ heteroaryl optionally
substituted with one or more RA or R1 substituents attached on either or both
the L and heteroaryl
groups, 63) -0-L-(N(R1)-L)0 ¨ heterocyclyl optionally substituted with one or
more RA or R1 substituents
attached on either or both the L and heterocyclyl groups, 64) -0-L-(N(R1)-08-
aryl optionally substituted
with one or more RA or R1 substituents, 65) -S-L- heteroaryl optionally
substituted with one or more RA
or R1 substituents, 66) -S-L- heterocyclyl optionally substituted with one or
more RA or R1 substituents,
67) -S-L- aryl optionally substituted with one or more RA or R1 substituents
attached on either or both
the L and aryl groups, 68) -S-L)2 NR1, 69) -S-L-(N(R1)-L)7 ¨ N(R1)R1, 70) -S-L-
(N(R1)-L)n ¨ heteroaryl
optionally substituted with one or more RA substituents, 71) -S-L-(N(R1)-L)8 ¨
heterocyclyl optionally
substituted with one or more RA substituents, 72) -S-L-(N(R1)-L)8 ¨ aryl
optionally substituted with one
or more RA substituents, 73) -NR1(R1), 74) -(N(R1)-L)0 ¨ N(R1)R1, 75) -
N(R1)L)2 ¨NR1, 76) -(N(R1)-L)8 ¨
N(R1)RA, 77) -(N(R1)-L)0 ¨ heteroaryl optionally substituted with one or more
RA or R1 substituents, 78)-
(N(R1)-L)2 ¨ heterocyclyl optionally substituted with one or more RA or R1
substituents, 79) -(N(R1)-L)8 ¨
aryl optionally substituted with one or more RA or R1 substituents, 80) -
heteroaryl optionally substituted
with one or more RA substituents, or 81) -aryl optionally substituted with one
or more RA substituents.
In this list, each substituent is optionally attached to the L group if it is
not already present; and when
two R1 substituents are present on the same nitrogen atom, then each R1
substituent is independently
selected from the list of R1 values described thereafter; and n is an integer
equal to either 0, 1, 2, 3, 4,
or 5; and, when (R1) and R1 are attached to a nitrogen atom, optionally they
join together with the
nitrogen atom to form a 3 to 7-membered ring which optionally includes one or
more other heteroatom
selected from N, 0 and S, optionally the ring is substituted with one or more
R1 or RA.
[0035] L is: 1) -C1-6 alkyl, 2) -C2-6 alkenyl, 3) -C2-6 alkynyl, 4) -C3-7
cycloalkyl, 5) -03-7 cycloalkenyl, 6)
heterocyclyl, 7) -C1_6alkyl-C3_7 cycloalkyl, 8) -01_6alkyl-heterocyclyl, 9)
aryl, or 10) heteroaryl. In this list,
11
the alkyl, the alkenyl, the alkynyl, the cycloalkyl, the cycloalkenyl, the
heterocyclyl, the aryl and the
heteroaryl groups are each independently optionally substituted with one or
two RA substituent.
[0036] R1 is: 1) -H, 2) -C1i6 alkyl, 3) -C2-6 alkenyl, 4) -C2.6 alkynyl, 5) -
C3-7 cycloalkyl, 6) -C3_7
cycloalkenyl, 7) -C1-6 perfluorinated, 8) -heterocyclyl, 9) -aryl, 10) -
heteroaryl, 11) -benzyl, or 12) 5-
R3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanoyl. In
this list, the alkyl, the
alkenyl, the alkynyl, the cycloalkenyl, the perfluorinated alkyl, the
heterocyclyl, the aryl, the heteroaryl
and the benzyl groups are each independently optionally substituted with 1, 2
or 3 RA or R1
substituents.
[0037] R2 is: 1) -H, 2) -01_6 alkyl, 3) -SR1, 4) -C(0)R1, 5) -S(0)R1, 6) -
S(0)2R1, 7) -benzyl optionally
substituted with 1, 2 or 3 RA or RI substituents, 8) -L-heteroaryl optionally
substituted with one or more
RA or R1 substituents attached on either one or both the L and the heteroaryl
groups, 9) -L-heterocyclyl
optionally substituted with one or more RA or RI substituents attached on
either one or both the L and
the heterocyclyl groups, 10) -L-aryl optionally substituted with one or more
RA or R1 substituents
attached on either one or both the L and the aryl groups, 11) -heteroaryl
optionally substituted with one
or more RA or R1 substituents, or 12) -aryl optionally substituted with one or
more RA or RI substituents.
In this list, each substituent is optionally attached to the L group if it is
not already present.
[0038] RA is: 1) -halogen, 2) -CF3, 3) -OH, 4) -0R1, 5) -L-OH, 6) -LORI, 7) -
0CF3, 8) -SH, 9) -SR1, 10)
-CN, 11) -NO2, 12) -NH2, 13) -NHRI, 14) -NR1R1, 15) -L-NH2, 16) -L-NHRI, 17) -
L-NR4R1, 18) -L-SRI,
19) -L-S(0)R1, 20) -L-S(0)2R1, 21) -C(0)0H, 22) -C(0)0R1, 23) -C(0)NH2, 24) -
C(0)NHR1, 25) -
C(0)N(R1)R1, 26) -NHC(0)R1, 27) -NR1C(0)R1, 28) -NHC(0)0R1, 29) -NR1C(0)0R1,
30) -0C(0)NH2,
31) -0C(0)NHR1, 32) -0C(0)N(R1)R1, 33) -0C(0)R1, 34) -C(0)R1, 35) -NHC(0)NH2,
36) -
NHC(0)NHR1, 37) -NHC(0)N(R1)R1, 38) -NR1C(0)NH2, 39) -NR1C(0)NHR1, 40) -
NR1C(0)N(R1)R1, 41)
-NHS(0)2R1, 42) -NR1S(0)2R1, 43) -S(0)2NH2, 44) -S(0)2NHR1, 45) -S(0)2N(R1)R1,
46) -S(0)R1, 47) -
S(0)2R1, 48) -0S(0)2R1, 49) -S(0)20R1, 50) -benzyl, 51) -N3, or 52) -C(-N-41-
)(CF3). In this list, the
benzyl group is optionally substituted with 1, 2 or 3 RA or RI substituents
In one embodiment, there is provided a compound of general formula Ill or IV,
CA 2862140 2019-01-23
1 1 a
I N z N
I
-N -N
R4R3N (L-Xi
III iv
or a salt thereof,
wherein Z and R2 are each as defined above, and m is an integer from 1 to 6,
and wherein when m is 2 or more, X are the same or different and are each
independently NR1, CH2 ,
0 or S, wherein R1 is as defined above, and Li are the same or different and
are each independently L
as defined above,
and wherein R3 and R4 are the same or different and are each independently H,
Ri as defined above,
or they join together with N to form a 3 to 7-membered ring which optionally
includes one or more other
heteroatom selected from N, 0 and S, optionally the ring is substituted with
one or more R1 or RA.
[0039] In embodiments of the invention, the compounds have the general Formula
IIA, IIB, IIC, IVA or
VIA shown below. Salts or prodrugs of such compounds are also within the scope
of the compounds
according to the invention.
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12
0
R10
N
¨N
I IA
Het
N
R2
¨N
I I B
R5
R6
z N
¨N
'IC
N
¨N
N RI
NR3R4
IVA
N R1
NR3R4
VIA
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13
[0040] In embodiments of the invention according to compounds of general
formula IIA above, R1, W
and R2 are each as defined herein above.
[0041] In embodiments of the invention according to compounds of general
formula IIB above, W and
R2 are each as defined herein above, and Het is a 3 to 7-membered heterocycle,
optionally substituted
with one or more R1 or RA as defined herein above.
[0042] In embodiments of the invention according to compounds of general
formula IIC above, W and
R2 are each as herein above; R5 and R6 are the same or different and are each
independently L as
defined herein above, or they join together with C to form a 5 to 7-membered
ring which optionally
includes one or more heteroatom selected from N, 0 and S, and optionally the
ring is substituted with
one or more R1 or RA. In further embodiments, the ring is a 5-membered ring
and the heteroatom is a
nitrogen atom. Still in further embodiments, the ring includes four nitrogen
atoms. Still in further
embodiments, R2 is benzyl.
[0043] In embodiments of the invention according to compounds of general
formula IVA above, W, L,
R1 and R2 are each as defined herein above. Also, m, Li, R3 and R4 are each as
defined herein above.
[0044] In embodiments of the invention according to compounds of general
formula VIA above, Z is
CO2Me or 2-methyl-2H-tetrazol-5-y1; R2 is benzyl, 3-thienylmethyl or 3-
pyridinyl methyl; and W is NH-L-
N(R1)R1 wherein L is 024 alkyl and R1 is C14 alkyl or (R1) and R1 join
together with the nitrogen atom to
which they are attached to form a 3 to 7-membered ring, which optionally
includes one or more other
heteroatom selected from N, 0 and S, optionally the ring is substituted with
one or more R1 or RA.
[0045] In embodiments of the invention, the compounds of the invention are
compounds No. 1 to 55
depicted in Table 1 herein below. Salts or prodrugs of such compounds are also
within the scope of the
compounds according to the invention.
[0046] In further embodiments of the invention, the compounds have the
formulas depicted in Table 1
herein below. Salts or prodrugs of such compounds are also within the scope of
the compounds
according to the invention.
Definitions:
[0047] Unless otherwise specified, the following definitions apply:
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14
[0048] The singular forms "a", "an" and "the" include corresponding plural
references unless the
context clearly dictates otherwise.
[0049] As used herein, the term "comprising" is intended to mean that the list
of elements following the
word "comprising" are required or mandatory but that other elements are
optional and may or may not
be present.
[0050] As used herein, the term "consisting of" is intended to mean including
and limited to whatever
follows the phrase "consisting of." Thus the phrase "consisting of' indicates
that the listed elements are
required or mandatory and that no other elements may be present.
[0051] As used herein, the term "alkyl" is intended to include both branched
and straight chain
saturated aliphatic hydrocarbon groups having the specified number of carbon
atoms, for example, Ci-
06 in 01-06 alkyl is defined as including groups having 1, 2, 3, 4, 5 or 6
carbons in a linear or branched
saturated arrangement. Examples of Cl-CG alkyl as defined above include, but
are not limited to,
methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, i-butyl, pentyl, and
hexyl.
[0052] As used herein, the term "cycloalkyl" is intended to mean a monocyclic
saturated aliphatic
hydrocarbon group having the specified number of carbon atoms therein, for
example, 03-07 in 03-07
cycloalkyl is defined as including groups having 3, 4, 5, 6 or 7 carbons in a
nnonocyclic saturated
arrangement. Examples of 03-07 cycloalkyl as defined above include, but are
not limited to,
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.
[0053] As used herein, the term, "alkenyl" is intended to mean unsaturated
straight or branched chain
hydrocarbon groups having the specified number of carbon atoms therein, and in
which at least two of
the carbon atoms are bonded to each other by a double bond, and having either
E or Z regiochemistry
and combinations thereof. For example, 02-06 in 02-06 alkenyl is defined as
including groups having 2,
3, 4, 5 or 6 carbons in a linear or branched arrangement, at least two of the
carbon atoms being bonded
together by a double bond. Examples of 02-06 alkenyl include, but are not
limited to, ethenyl (vinyl), 1-
propenyl, 2-propenyl, 1-butenyl and the like.
[0054] As used herein, the term "alkynyl" is intended to mean unsaturated,
straight chain hydrocarbon
groups having the specified number of carbon atoms therein and in which at
least two carbon atoms are
bonded together by a triple bond. For example 02-04 alkynyl is defined as
including groups having 2, 3
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or 4 carbon atoms in a chain, at least two of the carbon atoms being bonded
together by a triple bond.
Examples of such alkynyl include, but are not limited to, ethynyl, 1-propynyl,
2-propynyl and the like.
[0055] As used herein, the term "cycloalkenyl" is intended to mean a
monocyclic saturated aliphatic
hydrocarbon group having the specified number of carbon atoms therein, for
example, C3-07 in C3-07
cycloalkenyl is defined as including groups having 3, 4, 5, 6 or 7 carbons in
a monocyclic arrangement.
Examples of C3-C7 cycloalkenyl as defined above include, but are not limited
to, cyclopentenyl,
cyclohexenyl and the like.
[0056] As used herein, the term "halo" or "halogen" is intended to mean
fluorine, chlorine, bromine or
iodine.
[0057] As used herein, the term "haloalkyl" is intended to mean an alkyl as
defined above, in which
each hydrogen atom may be successively replaced by a halogen atom. Examples of
haloalkyl include,
but are not limited to, CH2F, CHF2 and CF3.
[0058] As used herein, the term "aryl," either alone or in combination with
another radical, means a
carbocyclic aromatic monocyclic group containing 6 carbon atoms which may be
further fused to a
second 5- or 6-membered carbocyclic group which may be aromatic, saturated or
unsaturated.
Examples of aryl include, but are not limited to, phenyl, indanyl, 1-naphthyl,
2-naphthyl,
tetrahydronaphthyl and the like. The aryl may be connected to another group
either at a suitable
position on the cycloalkyl ring or the aromatic ring.
[0059] As used herein, the term "heteroaryl" is intended to mean a monocyclic
or bicyclic ring system
of up to 10 atoms, wherein at least one ring is aromatic, and contains from 1
to 4 hetero atoms selected
from the group consisting of 0, N, and S. The heteroaryl may be attached
either via a ring carbon atom
or one of the heteroatoms. Examples of
heteroaryl include, but are not limited to, thienyl,
benzimidazolyl, benzo[b]thienyl, fury!, benzofuranyl, pyranyl,
isobenzofuranyl, chromenyl, xanthenyl,
2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl,
pyridazinyl, indolizinyl,
isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl, 4H-quinolizinyl,
isoquinolyl, quinolyl, phthalazinyl,
napthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl,
isothiazolyl, isochromanyl, chromanyl,
isoxazolyl, furazanyl, indolinyl, isoindolinyl, thiazolo[4,5-N-pyridine,
tetrazolyl, oxadiazolyl, thiadiazolyl,
thienyl and fluoroscein derivatives.
16
[0060] As used herein, the term "heterocycle," "heterocyclic" or
"heterocycly1" is intended to mean a 3,
4, 5, 6, or 7 membered non-aromatic ring system containing from 1 to 4
heteroatoms selected from the
group consisting of 0, N and S. Examples of heterocycles include, but are not
limited to, pyrrolidinyl,
tetrahydrofuranyl, piperidyl, 3,5-dimethylpiperidyl, pyrrolinyl, piperazinyl,
imidazolidinyl, morpholinyl,
imidazolinyl, pyrazolidinyl, pyrazolinyl, and the like, where the attachment
to the ring can be on either
the nitrogen atom or a carbon atom of the ring such as described hereafter:
R'10
N ,_5sss ON." ON" N,scs
, and
CS-
[0061] As used herein, the term "optionally substituted with one or more
substituents" or its equivalent
term "optionally substituted with at least one substituent" is intended to
mean that the subsequently
described event of circumstances may or may not occur, and that the
description includes instances
where the event or circumstance occurs and instances in which it does not. The
definition is intended
to mean from zero to five substituents.
[0062] As used herein, the term "subject" or "patient" is intended to mean
humans and non-human
mammals such as primates, cats, dogs, swine, cattle, sheep, goats, horses,
rabbits, rats, mice and the
like.
[0063] If the substituents themselves are incompatible with the synthetic
methods described herein,
the substituent may be protected with a suitable protecting group (PG) that is
stable to the reaction
conditions used in these methods. The protecting group may be removed at a
suitable point in the reaction
sequence of the method to provide a desired intermediate or target compound.
Suitable protecting groups
and the methods for protecting and de-protecting different substituents using
such suitable protecting groups
are well known to those skilled in the art; examples of which may be found in
T. Greene and P. Wuts,
"Protecting Groups in Chemical Synthesis" (4th ed.), John Wiley & Sons, NY
(2007). Examples of protecting
groups used throughout include, but are not limited to, Fmoc, Bn, Boc, CBz and
COCF3. In some instances,
a substituent may be specifically selected to be reactive under the reaction
conditions used in the
methods described herein. Under these circumstances, the reaction conditions
convert the selected
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17
substituent into another substituent that is either useful in an intermediate
compound in the methods
described herein or is a desired substituent in a target compound.
[0064] As used herein, the term "pharmaceutically acceptable salt" is intended
to mean both acid and
base addition salts.
[0065] As used herein, the term "pharmaceutically acceptable acid addition
salt" is intended to mean
those salts which retain the biological effectiveness and properties of the
free bases, which are not
biologically or otherwise undesirable, and which are formed with inorganic
acids such as hydrochloric
acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the
like, and organic acids such as
acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic
acid, oxalic acid, maleic acid,
malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic
acid, cinnamic acid, mandelic
acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,
salicylic acid, and the like.
[0066] As used herein, the term "pharmaceutically acceptable base addition
salt" is intended to mean
those salts which retain the biological effectiveness and properties of the
free acids, which are not
biologically or otherwise undesirable. These salts are prepared from addition
of an inorganic base or
an organic base to the free acid. Salts derived from inorganic bases include,
but are not limited to, the
sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper,
manganese, aluminum
salts and the like. Salts derived from organic bases include, but are not
limited to, salts of primary,
secondary, and tertiary amines, substituted amines including naturally
occurring substituted amines,
cyclic amines and basic ion exchange resins, such as isopropylamine,
trimethylamine, diethylamine,
triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-
diethylaminoethanol,
dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine,
hydrabannine, choline, betaine,
ethylenediamine, glucosamine, methylglucamine, theobromine, purines,
piperazine, piperidine, N-
ethylpiperidine, polyamine resins and the like.
[0067] The compounds according to the invention or their pharmaceutically
acceptable salts may
contain one or more asymmetric centers, chiral axes and chiral planes and may
thus give rise to
enantiomers, diastereomers, and other stereoisomeric forms and may be defined
in terms of absolute
stereochemistry, such as (R)- or (S)- or, as (D)- or (L)- for amino acids. The
present is intended to
include all such possible isomers, as well as, their racennic and optically
pure forms. Optically active (+)
and (-), (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral
synthons or chiral reagents,
or resolved using conventional techniques, such as reverse phase HPLC. The
racemic mixtures may
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18
be prepared and thereafter separated into individual optical isomers or these
optical isomers may be
prepared by chiral synthesis. The enantiomers may be resolved by methods known
to those skilled in
the art, for example by formation of diastereoisomeric salts which may then be
separated by
crystallization, gas-liquid or liquid chromatography, selective reaction of
one enantiomer with an
enantiomer specific reagent. It will also be appreciated by those skilled in
the art that where the desired
enantiomer is converted into another chemical entity by a separation
technique, an additional step is
then required to form the desired enantiomeric form. Alternatively specific
enantiomers may be
synthesized by asymmetric synthesis using optically active reagents,
substrates, catalysts, or solvents
or by converting one enantiomer to another by asymmetric transformation.
[0068] Certain compounds according to the invention may exist as a mix of
epimers. Epimers means
diastereoisomers that have the opposite configuration at only one of two or
more stereogenic centers
present in the respective compound.
[0069] Compounds according to the invention may exist in Zwitterionic form and
the present includes
Zwitterionic forms of these compounds and mixtures thereof.
[0070] In addition, the compounds according to the invention also may exist in
hydrated and
anhydrous forms. Hydrates of the compound of any of the formulas described
herein are included. In a
further embodiment, the compound according to any of the formulas described
herein is a
monohydrate. In embodiments of the invention, the compounds described herein
comprise about 10%
or less, about 9 % or less, about 8% or less, about 7% or less, about 6% or
less, about 5% or less,
about 4% or less, about 3% or less, about 2% or less, about 1% or less, about
0.5% or less, about
0.1% or less by weight of water. In others embodiments, the compounds
described herein comprise,
about 0.1% or more, about 0.5% or more, about 1% or more, about 2% or more,
about 3% or more,
about 4% or more, about 5% or more, or about 6% or more by weight of water.
[0071] It may be convenient or desirable to prepare, purify, and/or handle the
compound in the form of
a prodrug. Thus, the term "prodrug", as used herein, pertains to a compound
which, when metabolized
(e.g., in vivo), yields the desired active compound. Typically, the prodrug is
inactive, or less active than
the desired active compound, but may provide advantageous handling,
administration, or metabolic
properties. Unless otherwise specified, a reference to a particular compound
also includes prodrugs
thereof.
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[0072] As used herein, the term "EC50" is intended to mean the concentration
that results in a 50%
increase in CD34+CD45RA- cell count compared to vehicle cultures (DMSO).
[0073] As used herein, the term "hematopoietic stem cells" or "HSCs" is
intended to mean cells having
both pluripotency which allows them to differentiate into functional mature
cells such as granulocytes
(e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes
(e.g., reticulocytes,
erythrocytes), thrombocytes (e.g., megakaryoblasts, platelet producing
megakaryocytes, platelets), and
monocytes (e.g., monocytes, macrophages), and the ability to regenerate while
maintaining their
pluripotency (self-renewal).
[0074] HSCs are part of the starting cell population. These cells are
optionally obtained from the body
or an organ of the body containing cells of hematopoietic origin. Such sources
include un-fractionated
bone marrow, umbilical cord, peripheral blood, liver, thymus, lymph and
spleen. All of the
aforementioned crude or un-fractionated blood products can be enriched for
cells having hematopoietic
stem cell characteristics in ways known to those of skill in the art.
[0075] As used herein, the term "starting cell population" is meant to
identify a cell population
comprising HSCs harvested from one of various sources mentioned above, as
known in the art. The
starting cell population can be enriched in CD34+ cells meaning a cell
population selected based on the
presence of the cell surface marker CD34+. 0D34+ cells can be detected and
counted using for
example flow cytometry and fluorescently labeled anti-CD34 antibodies.
Moreover, the starting cell
population may be used directly for expansion or frozen and stored for use at
a later point in time.
[0076] During hematopoiesis, HSCs first diverge into the progenitor stage into
the myeloid lineage and
the lymphoid lineage, then differentiate into myeloid stem cells (mixed colony
forming cells, CFU-
GEMM) and into lymphoid stem cells, respectively. Further, myeloid stem cells
differentiate into
erythrocytes via erythroid burst forming cells (BFU-E) and erythroid colony
forming cells (CFU-E), into
thrombocytes via megakaryocyte colony forming cells (CFU-MEG), into monocytes,
neutrophils and
basophils via granulocyte-macrophage colony forming cells (CFU-GM), and into
eosinophils via
eosinophil colony forming cells (CFU-Eo), while lymphoid stem cells
differentiate into T cells via T
lymphoid progenitor cells and into B cells via B lymphoid progenitor cells.
These myeloid stem cells and
various hematopoietic progenitor cells derived from them are identified by the
properties of colonies
they form on soft agar, semisolid methylcellulose media or the like in the
presence of various cytokines.
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[0077] The present invention also includes use of a compound according to the
invention and as
defined herein, or a salt thereof, in the preparation of a medicament for the
treatment of a subject (or
patient) suffering from the following non-limiting list of disorders:
autologous or allogeneic
transplantation or treatment of a subject (or patient) suffering from the
above-mentioned disorders or
from auto-immune disorders. Examples of hematological malignancies/disorders
and congenital
diseases may include, without limitation, acute myeloid leukemia, acute
lymphoblastic leukemia,
chronic myeloid leukemia, chronic lymphocytic leukemia, myeloproliferative
disorders, myelodysplastic
syndromes, multiple myeloma, non-Hodgkin's lymphoma, Hodgkin's disease,
aplastic anemia, pure red
cell aplasia, hemoglobinuria, Fanconi anemia, thalassemia, sickle cell anemia,
Wiskott-Aldrich
syndrome, inborn errors of metabolism (such as Gaucher disease among others).
Examples of
immunological disorders that may benefit from transplantation are numerous and
include multiple
sclerosis, lupus, certain forms or arthritis, severe combined
immunodeficiencies, and the like.
[0078] Thus, the present invention encompasses administration, to a patient
suffering from any one of
the above-mentioned disorders/malignancies, HSCs that are expanded using a
compound according to
the invention.
[0079] Furthermore, the compounds and compositions as described can be used in
the following non-
limiting settings: autologous or allogeneic transplantation or treatment of a
subject (or patient) suffering
from the above-mentioned disorders or from auto-immune disorders. Examples of
hematological
malignancies/disorders and congenital diseases may include, without
limitation, acute myeloid
leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic
lymphocytic leukemia,
myeloproliferative disorders, myelodysplastic syndromes, multiple myeloma, non-
Hodgkin's lymphoma,
Hodgkin's disease, aplastic anemia, pure red cell aplasia, hemoglobinuria,
Fanconi anemia,
thalassemia, sickle cell anemia, Wiskott-Aldrich syndrome, inborn errors of
metabolism (such as
Gaucher disease among others). Examples of immunological disorders that may
benefit from
transplantation are numerous and include multiple sclerosis, lupus, certain
forms or arthritis, severe
combined immunodeficiencies, and the like.
[0080] Thus, the present invention encompasses administration, to a patient
suffering from any one of
the above-mentioned disorders/malignancies, HSCs that are expanded using a
compound according to
the invention.
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[0081] Also encompassed within the present invention is a cell population
obtained after expansion
using the method according to the invention and as described herein. Both
hematopoietic stem and
progenitor cells can be harvested from adult, umbilical cord blood, fetal or
embryonic sources. Cell
expansion using the method of the present invention can lead to an increase in
the number of
progenitor cells which is useful in hastening the time to neutrophil or
platelet engraftment, for example.
Such method comprises: culturing a starting population comprising HSCs with an
agent capable of
increasing the number of HSCs. The starting population may be enriched in the
cell surface marker of
interest or a combination thereof (for e.g. CD34+, CD34+CD45RA+/-)
[0082] Methods for expanding HSCs
[0083] The invention therefore relates to a method for expanding hematopoietic
stem cells, comprising
(a) providing a starting cell population comprising hematopoietic stem cells
and (b) culturing said
starting cell population ex vivo under suitable conditions for expanding
hematopoietic stem cells.
[0084] The invention therefore relates to a method for expanding hematopoietic
stem cells, comprising
(a) providing a starting cell population comprising hematopoietic stem cells
and (b) culturing said
starting cell population ex vivo under suitable conditions for expanding
hematopoietic stem cells.
[0085] In one specific embodiment, said method for expanding hematopoietic
stem cells, comprises
(a) providing a starting cell population comprising hematopoietic stem cells
and (b) culturing said
starting cell population ex vivo in the presence of the compound or
composition of the present
invention.
[0086] The cell population may first be subjected to enrichment or
purification steps, including
negative and/or positive selection of cells based on specific cellular markers
in order to provide the
starting cell population. Methods for isolating said starting cell population
based on specific cellular
markers may use fluorescent activated cell sorting (FACS) technology also
called flow cytometry or
solid or insoluble substrate to which is bound antibodies or ligands that
interact with specific cell surface
markers. For example, cells may be contacted with a solid substrate (e.g.,
column of beads, flasks,
magnetic particles) containing the antibodies and any unbound cells are
removed. When a solid
substrate comprising magnetic or paramagnetic beads is used, cells bound to
the beads can be readily
isolated by a magnetic separator.
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[0087] In one embodiment, said starting cell population is enriched in CD34+
cells. Methods for
enriching blood cell population in CD34+ cells include kits commercialized by
Miltenyi Biotec (CD34+
direct isolation kit, Miltenyi Biotec, Bergisch, Gladbach, Germany) or by
Baxter (lsolex 3000).
[0088] The amount of cord blood from a single birth is often inadequate to
treat an adult or an older
child. One advantage of the expansion method using the compound or composition
of the invention is
that it enables the production of a sufficient amount of hematopoietic stem
cells from only one cord
blood unit.
[0089] Accordingly, in one embodiment, the starting cell population is derived
from neonatal umbilical
cord blood cells which have been enriched in CD34+ cells. In one related
embodiment, said starting cell
population is derived from one or two umbilical cord blood units.
[0090] In another embodiment, the starting cell population is derived from
human mobilized peripheral
blood cells which have been enriched in CD34+ cells. In one related
embodiment, said starting cell
population is derived from human mobilized peripheral blood cells isolated
from only one patient.
[0091] Said starting cell population may preferably contain at least 50% CD34+
cells, in some
embodiments, more than 90% of CD34+ cells.
[0092] Culture conditions of the starting cell population for hematopoietic
stem cell expansion will vary
depending on the starting cell population, the desired final number of cells,
and desired final proportion
of HSCs.
[0093] In one specific embodiment, in particular, using a starting cell
population from umbilical cord
blood cells enriched in CD34+ cells, the culturing conditions comprises the
use of other cell expanding
factors like cytokines and growth factors, generally known in the art for HSC
expansion. Such cytokines
and growth factors can be biologics or small molecules and they include
without limitation IL-1, IL-3, IL-
6, IL-11, G-CSF, GM-CSF, SCF, FIT3-L, thrombopoietin (TP0), erythropoietin,
and analogs thereof. As
used herein, 'analogs include any structural variants of the cytokines and
growth factors having the
biological activity as the naturally occurring forms, including without
limitation, variants with enhanced or
decreased biological activity when compared to the naturally occurring forms
or cytokine receptor
agonists such as an agonist antibody against the TPO receptor (for example,
VB22B sc(Fv)2 as
detailed in patent publication WO 2007/145227, and the like). Cytokine and
growth factor combinations
are chosen to expand HSC and progenitor cells while limiting the production of
terminally differentiated
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23
cells. In one specific embodiment, one or more cytokines and growth factors
are selected from the
group consisting of SCF, Flt3-L and TPO.
[0094] Human IL6 or interleukin-6, also known as B-cell stimulatory factor 2
has been described by
(Kishimoto, Ann. review of 1 mm. 23:1 2005) and is commercially available.
Human SCF or stem cell
factor, also known as c-kit ligand, mast cell growth factor or Steel factor
has been described (Smith, M
A et al., ACTA Haematologica, 105, 3:143, 2001) and is commercially available.
Flt3-L or FLT-3 Ligand,
also referred as FL is a factor that binds to flt3-receptor. It has been
described (Hannum C, Nature 368
(6472): 643-8) and is commercially available. TPO or thrombopoietin, also
known as megakarayocyte
growth factor (MGDF) or c-Mpl ligand has been described (Kaushansky K (2006).
N. Engl. J. Med. 354
(19): 2034-45) and is commercially available.
[0095] The chemical components and biological components mentioned above may
be used not only
by adding them to the medium but also by immobilizing them onto the surface of
the substrate or
support used for the culture, specifically speaking, by dissolving a component
to be used in an
appropriate solvent, coating the substrate or support with the resulting
solution and then washing away
an excess of the component. Such a component to be used may be added to the
substrate or support
preliminarily coated with a substance which binds to the component.
[0096] The expansion of HSC may be carried out in natural medium, a semi-
synthetic medium or a
synthetic medium in terms of composition, and may be a solid medium, a
semisolid medium or a liquid
medium in terms of shape, and any nutrient medium used for hematopoietic stem
cell and/or
hematopoietic progenitor cell culture, which is supplemented with the mixtures
of cell expanding factors
described above. Such medium typically comprises sodium, potassium, calcium,
magnesium,
phosphorus, chlorine, amino acids, vitamins, cytokines, hormones, antibiotics,
serum, fatty acids,
saccharides or the like. In the culture, other chemical components or
biological components may be
incorporated singly or in combination, as the case requires. Such components
to be incorporated in the
medium may be fetal calf serum, human serum, horse serum, insulin,
transfferin, lactoferrin,
cholesterol, ethanolamine, sodium selenite, monothioglycerol, 2-
mercaptoethanol, bovine serum
albumin, sodium pyruvate, polyethylene glycol, various vitamins, various amino
acids, agar, agarose,
collagen, methylcellulose, various cytokines, various growth factors or the
like. Examples of such basal
medium appropriate for a method of expanding HSCs include, without limitation,
StemSpanTM Serum-
Free Expansion Medium (SFEM) (StemCell Technologies, Vancouver, Canada),
StemSpanTM H3000-
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24
Defined Medium (StemCell Technologies, Vancouver, Canada), CellGroTM, SCGM
(CellGenix, Freiburg
Germany), StemProTm-34 SFM (lnvitrogen), Dulbecco's Modified Eagles's Medium
(DMEM), Ham's
Nutrient Mixture H12 Mixture F12, McCoy's 5A medium, Eagles's Minimum
Essential Medium (EMEM),
0,MEM medium (alpha Modified Eagles's Minimum Essential Medium), RPMI1640
medium, Isocove's
Modified Dulbecco's Medium (IMDM), StemPro34 (lnvitrogen), X-VIVO 10
(Cambrex), X-VIVO 15
(Cambrex) and Stemline II (Sigma-Aldrich).
[0097] In one embodiment, the compound or the composition of the invention is
administered during
the expansion method of said starting cell population under a concentration
appropriate for HSC
expansion. In one specific embodiment, said compound or composition is
administered at a
concentration comprised between 1 and 3000 nmol or for example between 1 and
100 nmol.
[0098] In one specific embodiment where starting cell population essentially
consists of CD34+
enriched cells from one or two cord blood units, or from mobilized PB cells or
from harvested bone
marrow, the cells are grown under conditions for HSC expansion, for example
between 2 and 21 days
and/or until the indicated fold expansion and the characteristic cell
populations are obtained. In one
specific embodiment, the cells are grown ex vivo under conditions for HSC
expansion not more than 21
days, 12 days, 10 days or 7 days.
[0099] The cell population may then be washed to remove the compound or
composition of invention
and/or any other component of the cell culture and resuspended in an
appropriate cell suspension
medium for short term use or in a long-term storage medium, for example a
medium suitable for
cryopreservation.
[0100] The HSCs and/or hematopoietic progenitor cells can be cultured in a
culture vessel generally
used for animal cell culture such as a Petri dish, a flask, a plastic bag, a
TeflonTm bag, optionally after
preliminary coating with an extracellular matrix or a cell adhesion molecule.
The material for such a
coating may be collagens Ito XIX, fibronectin, vitronectin, laminins 1 to 12,
nitrogen, tenascin,
thrombospondin, von Willebrand factor, osteoponin, fibrinogen, various
elastins, various proteoglycans,
various cadherins, desmocolin, desmoglein, various integrins, E-selectin, P-
selectin, L-selectin,
immunoglobulin superfamily, Matrigel, poly-D-lysine, poly-L-lysine, chitin,
chitosan, Sepharose, alginic
acid gel, hydrogel or a fragment thereof. Such a coating material may be a
recombinant material having
an artificially modified amino acid sequence. The hematopoietic stem cells
and/or hematopoietic
progenitor cells may be cultured by using a bioreactor which can mechanically
control the medium
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composition, pH and the like and obtain high density culture (Schwartz R M,
Proc. Natl. Acad. Sci.
U.S.A., 88:6760, 1991; Koller M R, Bone Marrow Transplant, 21:653, 1998;
Koller, M R, Blood, 82: 378,
1993; Astori G, Bone Marrow Transplant, 35: 1101, 2005).
[0101] The invention further provides a cell population with expanded HSCs,
obtainable or obtained by
the expansion method described above. In one specific embodiment, such cell
population is
resuspended in a pharmaceutically acceptable medium suitable for
administration to a mammalian
host, thereby providing a therapeutic composition.
[0102] The invention further provides the cell population with expanded HSCs
or its composition for
use in allogeneic or autologous stem cell transplantation in a mammalian
subject.
[0103] The subject referred to herein is, for example, a bone marrow donor or
an individual with or at
risk for depleted or limited blood cell levels. Optionally, the subject is a
bone marrow donor prior to bone
marrow harvesting or a bone marrow donor after bone marrow harvesting. The
subject is optionally a
recipient of a bone marrow transplant. The methods described herein are
particularly useful in subjects
that have limited bone marrow reserve such as elderly subjects or subjects
previously exposed to an
immune depleting treatment or myeloablative treatment such as chemotherapy,
e.g., for treating
leukemia or lymphomas. The subject, optionally, has a decreased blood cell
level or is at risk for
developing a decreased blood cell level as compared to a control blood cell
level. As used herein the
term control blood cell level refers to an average level of blood cells in a
subject prior to or in the
substantial absence of an event that changes blood cell levels in the subject.
An event that changes
blood cell levels in a subject includes, for example, anemia, trauma,
chemotherapy, bone marrow
transplant and radiation therapy. For example, the subject has anemia or blood
loss due to, for
example, trauma.
[0104] The transplant may be a composition containing a buffer solution, an
antibiotic, a
pharmaceutical in addition to hematopoietic stem cells and/or hematopoietic
progenitor cells expanded
by the method of the present invention.
[0105] The expanded HSC population or the composition comprising the cell
population with
expanded HSCs is administered to the subject, for example, before, at the same
time, or after
chemotherapy, radiation therapy or a bone marrow transplant. The subject
optionally has depleted bone
marrow related to, for example, congenital, genetic or acquired syndrome
characterized by bone
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26
marrow loss or depleted bone marrow. Thus, the subject is optionally a subject
in need of
hematopoiesis. Optionally, the subject is a bone marrow donor or is a subject
with or at risk for depleted
bone marrow.
[0106] Hematopoietic stem cell manipulation is useful as a supplemental
treatment to chemotherapy
or radiation therapy. For example, HSCs are localized into the peripheral
blood and then isolated from a
subject that will undergo chemotherapy, and after the therapy the cells are
returned. Thus, the subject
is a subject undergoing or expected to undergo an immune cell depleting
treatment such as
chemotherapy, radiation therapy or serving as a donor for a bone marrow
transplant. Bone marrow is
one of the most prolific tissues in the body and is therefore often the organ
that is initially damaged by
chemotherapy drugs and radiation. The result is that blood cell production is
rapidly destroyed during
chemotherapy or radiation treatment, and chemotherapy or radiation must be
terminated to allow the
hematopoietic system to replenish the blood cell supplies before a patient is
re-treated with
chemotherapy. Therefore, as described herein, HSCs or blood cells made by the
methods described
herein are optionally administered to such subjects in need of additional
blood cells.
[0107] Provided are HSCs expanded by a compound or a composition of the
invention as described
above in combination with a therapeutic capable of enhancing the proliferation
of HSCs in vivo, in vitro,
or ex vivo (for example, a small molecule, an antibody, or the like) and
optionally at least one
pharmaceutically acceptable excipient or carrier. By a therapeutic capable of
enhancing HSC
proliferation is meant: an agonist antibody against the TPO receptor (for
example, VB22B sc(Fv)2 as
detailed in patent publication WO 2007/145227, and the like); a cytokine such
as SCF, IL-6, Flt-3
ligand, TPO or a TPO mimetic (for example, such as described in
W0/2007/022269; W0/2007/009120;
W0/2004/054515; W0/2003/103686; W0/20021085343; WO/2002/049413;
W0/2001/089457;
W0/2001/039773; WO/2001/034585; W0/2001/021180; W0/2001/021180;
W0/2001/017349;
WO/2000/066112; WO/2000/035446; WO/2000/028987; W0/2008/028645; and the like);
granulocyte
colony stimulating factor (G-CSF); granulyte macrophage colony stimulating
factor (GM-CSF); a
prostaglandin or a prostaglandin receptor agonist (for example, prostaglandin
E2 receptor-1 (EP-I)
agonist, prostaglandin E2 receptor-2 (EP-2) agonist, prostaglandin E2 receptor-
3 (EP-3) agonist and
prostaglandin E2 receptor-4 (EP-4) agonists, as detailed in patent publication
WO/2008/073748);
tetraethylenepentamine (TEPA); Notch-ligands (Delta-1); and/or a WNT agonist.
In addition, culturing
stem cells with mesenchymal stem cells (MSCs) prevents graft-versus-host
disease (GVHD) and may
help stem cell expansion.
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[0108] By pharmaceutically acceptable is meant a material that is not
biologically or otherwise
undesirable, i.e., the material may be administered to a subject or cell,
without causing undesirable
biological effects or interacting in a deleterious manner with the other
components of the
pharmaceutical composition in which it is contained. The carrier or excipient
is selected to minimize
degradation of the active ingredient and to minimize adverse side effects in
the subject or cell.
[0109] The compositions are formulated in any conventional manner for use in
the methods described
herein. Administration is via any route known to be effective by one of
ordinary skill. For example, the
composition is administered orally, parenterally (e.g., intravenously), by
intramuscular injection, by
intraperitoneal injection, transdermally, extracorporeally, intranasally or
topically.
[0110] The preferred method of administration is intravenous infusion. The
number of cells transfused
will take into consideration factors such as sex, age, weight, the types of
disease or disorder, stage of
the disorder, the percentage of the desired cells in the cell population and
the amount of cells needed to
produce a therapeutic benefit. In one particular embodiment, the composition
is administered by
intravenous infusion and comprises at least 0.3 x 105 CD344/ kg or > 2 x 106
CD34+ for cord blood
and 2.5 x 105 CD344/ kg or more for bone marrow or mobilized peripheral blood
cells. In one specific
embodiment, the infused cells are all deriving from expanded cord blood cells
from a single birth.
[0111] Expanded hematopoietic stem cells and/or hematopoietic progenitor cells
may be infused by
drip, for example, in the case of treatment of leukemia, into patients
pretreated with an anticancer drug,
total body irradiation or an immunosuppressive drug for eradication of cancer
cells or for facilitation of
donor cell engraftment. The disease to be treated, the pretreatment and the
cell transplantation method
are selected appropriately by the person in charge. The engraftment of so
transplanted hematopoietic
stem cells and/or hematopoietic progenitor cells in the recipient, the
recovery of hematopoiesis, the
presence of side effects of the transplantation and the therapeutic effect of
the transplantation can be
judged by an ordinary assay used in transplantation therapy.
[0112] As described above, the present invention makes it possible to expand
hematopoietic stem
cells and/or hematopoietic progenitor cells and to carryout transplantation
therapy safely and easily in a
short term by using the expanded HSCs.
[0113] Also provided herein is a kit comprising one or more containers filled
with one or more of the
ingredients described herein. Such kits optionally comprise solutions and
buffers as needed or desired.
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The kit optionally includes an expanded population of stem cells made by the
methods described above
or can contain containers or compositions for making an expanded population of
HSCs. In particular,
the invention provides a kit for expanding ex vivo hematopoietic stem cells,
comprising a compound as
defined in the Summary of Invention and instructions for use of such compound
in a method for HSC
expansion and, optionally, one or more cell expanding factors, or media for
cell growth, in particular
media for HSC growth as described above. The kit may further comprise
antibodies for monitoring
production of the cells, such as anti-CD34, anti-0D38 and/or anti-CD45RA
antibodies. In one specific
embodiment, such kit further includes one or more cell expanding factor
selected from the group
consisting of IL6, FLT3-L, SCF and TPO. Optionally associated with such
pack(s) or kit(s) are
instructions for use.
[0114] In vivo application: Also provided is a kit for providing an effective
amount of a compound of the
invention to increase HSCs in a subject comprising one or more doses of the
compound for use over a
period of time, wherein the total number of doses of the compound of the
invention in the kit equals the
effective amount sufficient to increase HSCs in a subject. The period of time
is from about one to
several days or weeks or months. Thus, the period of time is from at least
about 5, 6, 7, 8, 10, 12, 14,
20, 21, 30 or 60 days or more or any number of days between one and 180.
Biological Assays
Screening Assay:
[0115] To identify novel putative agonists of HSC self-renewal, we have
adapted a high throughput
based-screening assay to test a library of small molecule compounds (5,280 low
molecular weight
compounds) on primary human mobilized CD34+ cells. It is to be understood that
the same approach
applies to CD34+ cells from the various sources known by a person skilled in
the art to isolate CD34+
cells. Mononuclear cells were stained with Mouse antihuman CD34+APC (from BD
Pharmingen) and
subsequently magnetically labeled with Anti-APC magnetic MicroBeads (from
MACS, Miltenyi Biotec).
The magnetically labeled cells were retained using AutoMACS column. Our study
was based on the
fact that mobilized peripheral blood-derived CD34+CD45RA- cells cultured in
media supplemented with
interleukin-6, thrombopoietin, Flt-3 ligand, and stem cell factor, would
promote the expansion of
mononuclear cells (MNC) concomitant with a decrease in CD34+CD45RA- population
and HSC
depletion. Low molecular weight compounds preventing this loss could therefore
act as agonists of
HSC expansion.
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[0116] In a 384-well plate, 2000 CD34+cells/well were cultured in 50 pl medium
containing 1 pM of test
compounds or 0.1% DMSO (vehicle). The proportion of CD341-CD45RA- cells was
determined at the
start of the experiment and after a 7-day incubation. Six out of the 5,280
compounds of different
chemical backgrounds initially tested promoted CD34+CD45RA- cell expansion,
and seventeen (17)
enhanced differentiation as determined by the increase in proportions of 0D34-
CD45RA+ cells
compared to control (DMSO). The six compounds promoting expansion of the
CD34+CD45RA- cell
population were re-analyzed in a secondary screen. Four out of these six
compounds act as aryl
hydrocarbon receptor (AhR) antagonists, a mechanism of action (same as the SRI
s) shown to
promote the ex vivo expansion of huCD34+ cells. The remaining two compounds,
determined as not
being aryl hydrocarbon receptor (AhR) antagonists, were shown to promote the
expansion of MNCs
including CD34+ cells during 7-day incubation. One of those two remaining
compounds identified is
Compound 1 (Table 1).
[0117] The following biological assays were used to assess the effect of the
compounds of the
invention on hematopoietic stem cell expansion. Culture medium: The culture
medium used consisted
of serum-free medium supplemented with the following recombinant cytokines.
interleukin-6,
thrombopoietin, Flt-3 ligand, and stem cell factor, each at a final
concentration of 10Ong/ml, in the
presence of vehicle (DMSO), positive control (SRI), or compound of the
invention or a combination of
compounds. Cell Culture: CD34+ cell purity of initial harvests was higher than
90%, as determined by
flow cytometry. The CD34+CD45RA- subpopulation reached purity levels higher
than 70%. Cells were
plated at 40,000 cells/ml and incubated for 7 to 12-days at 37 C in 5% CO2,
For long term cultures,
200,000 CD34+cells/m1 from mobilized PB were plated with serum free media
supplemented with
interleukin-6, thrombopoietin, Flt3 ligand, and stem cell factor, each at a
final concentration of 10Ong/ml,
in the presence of vehicle (DMSO), positive control, or a compound of the
invention at 500 nM. After a
10-day ex vivo culture Compound 1 (Table 1) promoted more than 7-fold
expansion of MNCs, more
than 5-fold increase in CD34+ cells over input values (day 0), and almost 4-
fold increase over the
values determined for vehicle. After 10 days of ex vivo culture, Compound 1-
treated cells retained a
high level of 0D34 expression (65.8 5.5%) compared to cells cultured with
vehicle (DMSO)
(22.8 0.9%). Moreover,
only Compound 1-treated cells retained the highest expression of
CD34+CD45RA- population (24.8 0.9%) compared to that of vehicle (4.7 0.4%).
The numbers of
CD34+CD45RA- cultured with Compound 1 increased by almost 3-fold compared to
that of vehicle and
7-fold more than that of input. Finally, the compounds of the invention were
assayed in a dose-
response format (concentrations ranging from 1 nM to 5000 nM) to determine the
effective
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concentration that produced a 50% increase in the number of CD34+CD45RA- cells
compared to
vehicle condition. The results are shown in Table 1.
Compound 1 does not act through aryl hydrocarbon (AhR) pathway (Fig. 1)
[0118] We next documented that the impact of Compound 1 on primitive
CD34+CD45RA- primitive
hematopoietic cell is rapidly reversible in culture. This effect is best shown
in Figure 2 where CD34+
mobilized peripheral blood cells are cultured in the presence of Compound 1
for up to 7 days, at which
time the Compound1 is removed by washing the cells. The green dotted line
shows that the reduction
in the proportion of CD34+ CD45RA- cells rapidly follows that of controlled
cultures (DMSO: solid blue
and dotted lines) whereas cells maintained in the presence of Compound 1
retain a more primitive
phenotype throughout the 2 week culture (solid green line). These results
clearly indicate that within 2
days of compound-free exposure, cells have already acquired differentiation
markers as seen in control
cultures. Thus, the impact of Compound 1 is rapidly reversible on primitive
human cells.
Compound 1 is not a mitogen (Fig. 3)
[0119] We also showed that Compound 1 does not independently trigger cell
proliferation in the
absence of growth factor. These results, shown in Figure 3, indicate that
similar to what was observed
with an antagonist of Aryl Hydrocarbon receptor (SRI), Compound 1 was not able
to induce cell
proliferation in the absence of any of the 3 listed growth factors, namely
Flt3, TPO and SCF. This
indicate that akin to SRI, Compound l's effect on maintenance of primitive HSC
phenotype ex vivo is
not due to a mitogenic effect on this population but rather on the prevention
of cell diffentiation.
Compound 1 and Compound 40 both prevent cell differentiation and synergizes
with AhR antagonist
(Fig. 4)
[0120] We also assessed the impact of Compound 1 and also one of its potent
derivative Compound
on cell differentiation using both cytological analysis and flow cytometry.
Both types of studies
showed that Compound 1 and its derivative Compound 40 prevent cell
differentiation. FACS results are
shown in Figure 4. Figure 4A illustrates the impact of Compound 1 on mobilized
peripheral blood cell
differentiation. Following a 7 day expansion, a relatively pure (>85% CD34+)
population of mobilized
peripheral blood was exposed to control the (DMSO) or Compound 1 or SRI or
Compound 1 + SRI.
Results strongly indicate that cells rapidly loose CD34 cell surface
expression in control cultures
whereas this effect is partly abrogated by introducing optimal levels of SRI
and Compound I.
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31
Interestingly both SRI and Compound 1 synergize in maintaining CD34 expression
on the cell surface.
These observations have been repeated for cord blood specimens in Figure 4B
and also with
Compound 40 in Figure 4C.
[0121] In addition to this, we showed that the impact of Compound 1 or
Compound 40 is most
impressive on cells which have a more primitive phenotype. For example,
CD34+CD45RA- cells (upper
left quadrant in bottom panels of Figure 4A to 4C) are more numerous in
cultures supplemented with
Compound 1 or 40 than they are in SRI or in control cultures. Again additive
effects of Compound 1 or
compound 40 plus SRI is observed in these cultures.
[0122] Figure 4D provides a dose-response curve which indicates the potency of
Compound 40 in
preventing the disappearance of the CD34 marker on the surface of primitive
human HSC enriched
population. Note expression of CD34 varying with different dose of the
compound.
Compounds 1 and 40 expand human HSC phenotype ex vivo (Fig. 5)
[0123] Figure 5 shows that Compound 1 but also Compound 40 expand human HSC
phenotype ex
vivo in both short term and long term cultures. Figure 5A shows that total
cell count is increased by
about 20 fold above the input in cultures initiated with CD34+ mobilized
peripheral blood (mPB) and
maintained for 12-days. The level of expansion is the same whether cultures
are initiated with
Compound 1, SRI, Compound 1 + SRI, or control DMSO. Most strikingly the impact
of Compound 1 is
observed on more primitive CD34+CD45RA- cell subpopulation which expands by
about ten fold in the
presence of Compound 1 and by about fifteen fold in the presence of Compound 1
+ SRI. This
observation together with the results presented in Figure 4 strongly suggest
that Compound 1 has no
impact on cell proliferation but rather on preventing differentiation of CD34
+ CD45RA- cells leading to
their net expansion at the expense of more mature cells. Results in Figure 5B
indicate that Compound
1 will lead to a 30-40-fold expansion of CD341-CD45RA- cord blood cells over a
seven day period
whereas these cells are expanded by about fifteen fold in the presence of DMSO
(control). Figure 5C
shows similar results but this time with cultures extended to twelve days and
Compound 1 is replaced
by Compound 40. Again, as indicated in the left panel, total cell expansion is
the same whether cells
are exposed to DMSO (control), SR1, Compound 40 or both Compound 40 plus SRI.
Most
impressively, the more primitive CD34 + CD45RA- cells expand by 80-fold over
12-days in cultures
supplemented with Compound 40, whereas these cells expand a little less than
20- fold in cultures
initiated with SRI, showing the superiority of this compound over Aryl
Hydrocarbon Repressor
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32
antagonist.
[0124] In summary, these results show that Compound 1 and Compound 40 have
major effects on
expansion of CD34-' and the more primitive CD34+ CD45RA- population, both with
CD34+ derived from
mobilized peripheral blood or cord blood cells.
[0125] The ex vivo functionality of expanded cells was tested using the
conventional colony-forming
units in culture (CFU-C) assay. Untreated cells or cells incubated with DMSO,
positive control or a
compound of the invention were plated in methylcellulose medium in
conventional conditions. As an
example, Compound 1 (Table 1, Example 1) expands the number of multipotent
hematopoietic
progenitors. Methylcellulose culture of 1000 CD34+ mPB cells treated with
Compound 1 for 10 days
resulted in a 5-fold increase in multilineage granulocyte erythrocyte,
macrophage and megakaryocyte
(GEMM colonies) over input cells and a 10-fold increase compared to control
cells. This suggests that
Compound1 described herein also promotes expansion of multipotent progenitor
cells.
[0126] Impact of Compound 1 on cultured mPB HSC assessed using NSG mouse model
(Fig. 6)
[0127] We next assessed the impact of Compound 1 and Compound 40 on cord blood
and mobilized
peripheral blood human HSC expanded in vitro for ten to twelve days and
introduced in vivo in NSG
mouse model. The goal of these experiments is to verify that the impact of our
compound on primitive
HSC phenotype shown in Figures 4 and 5 is also observed on the long term
repopulating bone marrow
stem/progenitor cells. Figure 6 shows reconstitution of mouse bone marrow by
human cells assessed
thirteen weeks after transplantation. For mobilized blood, the outcome of
50,000 and 500,000 cells is
presented in 6A. As shown there, HSC agonist SRI was consistently better than
DMSO (control) in
expanding human stem cells as assessed in the NSG mouse model. Again Compound
1 appears to be
superior to SRI in these experiments. As seen in the in vitro cultures,
Compound 1 and SRI showed a
synergistic effect in these experiments (Fig. 6).
Impact of Compound 1 and 40 on cultured Cord Blood (CB) HSC assessed using NSG
mouse model
(Fig. 7)
[0128] Figure 7 shows the impact of Compound 1 and Compound 40 on cultured
cord blood human
HSC assessed in vivo in NSG mouse model. Results in Figure 7A indicate that
Compound 1 has a
clear effect on reconstitution activity of human cells when compared to
control cultures. These
experiments were done in short term cultures i.e. 7 days. Most importantly,
Figure 7B indicates that
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Compound 40 has quite an important effect with average levels of
reconstitution at 10% compared to
2% for DMSO control when using 1500 CD34+ cells. The greater impact of
Compound 40 in this
experiment over Compound 1 (Figure 7A) is potentially due to the longer
culture period used in
experiments described in Figure 7B. More definitive in vivo experiments are
provided in the next
section using longer culture periods (12 to 16 days).
Impact of Compound 1 and 40 on cultured Cord Blood (CB) HSC assessed using NSG
mouse model
(Fiq.8)
[0129] Zandstra et al. recently documented that a new method called fed-batch
optimizes in vitro
conditions leading to human HSC expansion (US Patent no. US7,795,024). We
wanted to verify
whether compounds of the invention are active in these previously optimized
fed-batch conditions. For
these studies, we assessed the expansion of cord blood-derived hematopoietic
stem cells (HSCs)
following in vitro culture with the fed-batch + Compound 40 for 12 or 16 days.
HSC numbers were
assessed based on engraftment of human cells into immune-deficient mice.
[0130] As shown in Figure 8 the addition of Compound 40 (Cpd 40) provided a
major effect on
expansion of all populations tested including CD34+CD45RA- cells up to at
least 16 days. This effect
was most impressive in the 12-16 day time points clearly demonstrating the
synergy between fed-batch
(FB in Fig. 8) and Compound 40.
[0131] Moreover, freshly enriched CD34+ cells and cells expanded for 12 or 16
days were
transplanted into female NOD/SCID/IL-2Ryc-null (NSG) mice, which had been sub-
lethally (250rad)
irradiated 24h prior to transplantation. Cells were injected intravenously
through the tail vein. At defined
time-points (week 3, week 9, and week 16), the animals were sacrificed and
bone marrow was
harvested from two tibias and two femurs. The bone marrow was depleted of red
blood cells and
assessed by flow cytometry to quantify the engraftment of human cells. Cells
were scored positive for
engraftment if .Ø5% of the cells were positive for human CD45 and human HLA-
ABC. At each time-
point of assessment, some animals were sent for independent histology
analysis.
[0132] As shown in Table 2 below, at the late 16-week time-point, there is a
dose response of
engraftment for each condition. Limiting dilution analysis revealed that the
highest HSC expansion was
produced by the fed-batch+Compound 40 12-day culture (18.4-fold). This was
significantly higher than
the expansion produced by the fed-batch control 12- day culture (8.1-fold).
Higher expansion was
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produced by both conditions with the 12-day culture as compared to the 16-day
culture. These results
provide unequivocal demonstration of net human HSC expansion in vitro in the
presence of Compound
40 and its activity in fed-batch conditions (Fig. 8 + Table 2).
Table 2: Summary of Week 16 limiting dilution engraftment data
frequency of INC day 0 HSC Net HSC
Condition HSCs expansion equivalent expansion*
Day 0 fresh 1/6115 NA 1/6115 NA
Day 12 Fed-batch 1/117676 156 1/754.3 8.1
Day 12 Fed-batch +
Cpd 40 1/54548 164 1/332.6 18.4
Day 16 Fed-batch 1/635952 413 1/1539.8 4
Day 16 Fed-batch +
Cpd 40 1/323585 495 1/653.7 9.4
[0133] * Net HSC Expansion = (# HSC Day 0)/(# HSC Day 12 or Day 16); measured
using limiting
dilution analysis (LDA) in NSG mice at week 16
SYNTHETIC METHODOLOGY
[0134] The synthetic methodology outlined below relates to embodiments of the
invention wherein
substituent Z is at the 7-position of the pyrimido indole nucleus. As will be
understood by a skilled
person, a similar synthetic methodology can be performed, with variations that
are apparent to such
person, for embodiments of the invention wherein substituent Z is at a
different position, such as for
example at the 5, 8 or 6-position, particularly at the 6-position.
[0135] Scheme 1 describes the synthesis of the common precursor (1-VI) to the
compounds of the
present invention. In the first step an aryl fluoride 1-1 is treated with an
alkyl cyanoacetate 1-11 in the
presence of a base such as, but not limited to, sodium hydride. The resulting
product 1-III is then
treated with a reducing agent such as, but not limited to, zinc dust in acetic
acid to provide amino
indoles 1-1V which are converted to the pyrimidines 1-V upon treatment with
formamide and ammonium
formate. Compounds 1-V are treated with reagents such as phosphoryl chloride
or phosphoryl bromide
to provide the reactive intermediates 1-V1 which are treated with amines 1-V11
to provide the
compounds 1-VIII of the present invention.
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Scheme 1
CN
Z 401 NO2 + LO Base Z NO2
Reducing
¨11. CN a ent
9
_,..
F OR
Acid
0 OR
1-I 1-11 1-III
H Z H
Z N N Activating
NH2 R2CO2Me N agent
_,...
OR (NHR) ¨N
0 HO
165 C 1-V
1-IV
Z = CO2R
H H
Z N Z N
heat
i ....._R2 +
¨N 1 -VII ¨N
X W
1-VI
X = CI, Br 1-VIII
[0136] Scheme 2 describes the preparation of compounds 2-V. The reaction of
4,6-dichloro-5-
iodopyrimidine 2-11 and the corresponding phenol, aniline or thiophenol 2-1
with or without a base
(Morsin M. et al. Chemistry-A European Journal, 2009, vol. 15, #6, pp. 1468-
1477) or a palladium
catalyst to give intermediates 2-111. The resulting intermediates 2-111 are
converted to the tricyclic adduct
2-1V with Pd(OAc)2 (Zhang M. et al. Tetrahedron Letters, 2002, vol.43, p.
8235). Finally, according to
Example 1 outlined herein below, the compounds of the present invention 2-V
are obtained.
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Scheme 2
Z 401 NH 2 CI N
N N
I a) fi b)
N I
,=1%1
CI CI CI
2-1V
2-1 2-11 2-111 c)
N N
a) heat-base; b) Pd(OAc)2; c) heat, W-Y N
2-V
[0137] Scheme 3: Compounds 4-11 are treated with hydroxyl amine followed by
dimethylacetamide
dimethylacetal (Tully W.R. et al. Journal of Medicinal Chemistry, 1991, vol.
34, p. 2060. This gives the
compounds 5-1.
Scheme 3
NC N
I 1 a) I
N
441
54
a) 1- NH2OH, 2- DMA acetal
[0138] Scheme 4: Intermediate 1B (Example 1 herein below) is treated with
propionitrile in
HCl/dioxane followed by a basic treatment to furnish methyl 2-ethy1-4-hydroxy-
9H-pyrimido[4,5-b]indole-
7-carboxylate (6-1). Then according to the procedures described for Example 1
compounds 6-11 are
obtained.
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Scheme 4
Me02C
Me02C N a) Me02C (
= / NH2 N I N
CO2Et
6-1 OH
Intermediate 1B 611
a) HCI, propionitrile; b) see Example 1
[0139] Scheme 5: Starting from methyl 3-fluoro-4-nitrobenzoate 7-land
according to the procedure of
Example 1, compounds 7-1I are obtained.
Scheme 5
NO2 N
a) fJJyN
-1
N
Me02C
Me02C
Af
7-1 7-11
a) see Example 1
Examples
General
[0140] Reported HPLC retention time are for reverse-phase HPLC (Agilent, 1200
series) using the
following conditions Solvent A: MeOH:H20:TFA (5:95:0.05); Solvent B:
MeOH:H20:TFA (95:5:0.05);
flow: 3.0 mL/min; gradient 0 to 100% B in 2.0 minutes; column: ZorbaxC18, 3.5
microns, 4.6 x 30 mm:
wavelength 220 nm.
[0141] Mass spectra were recorded on a 6210 G1969A LC/MSD TOF spectrometer
from Agilent
Technologies or on a Quadrupole LC/MS Model G6120B from Agilent Technologies
using the following
LC conditions: Solvent A: AcCN:H20:HCOOH (5:95:0.05); Solvent B:
AcCN:H20:HCOOH (95:5:0.05);
gradient 0 to 100% B in 2.0 minutes; flow: 0.3 mL / min; column: ZorbaxC18,
3.5 microns, 2.1 x 30 mm;
wavelength 220 nm.
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[0142] Experimental procedures
Example 1
0
¨N
HN
C)
Methyl 4-((3-(piperidin-111)propyl)amino)-9H-pyrimido[4,5-b]indole-7-
carboxylate
Intermediate 1A
NO2
N
0 0
methyl 4-(1-cyano-2-ethoxy-2-oxoethyl)-3-nitrobenzoate
[0143] Ethyl 2-cyanoacetate (10.9 mL, 102 mmol) was slowly added to a 60%
suspension of sodium
hydride (4.10 g, 102 mmol) in N,N-dimethylformamide (125 mL) at 0 C to give a
gray suspension. The
mixture was stirred at 0 C for 15 minutes and methyl 4-fluoro-3-nitrobenzoate
(10.2 g, 51 mmol) in N,N-
dimethylformamide (125 mL) was added. The resulting deep red mixture was
stirred at 0 C for 30
minutes and at room temperature for 3 hours. The reaction mixture was diluted
with IN HCI (40 mL)
and ethyl acetate (40 mL). The separated aqueous layer was extracted with
ethyl acetate (3 x 50 mL).
The organic layers were combined and dried over anhydrous sodium sulfate,
filtered and concentrated
to afford a residue (26 g) which was purified by flash-chromatography (started
with 100% hexanes and
gradually added ethyl acetate increments to complete with 100% ethyl acetate)
to afford 14.9 g of the
title compound. LCMS m/z 291.0 (M - H)-, retention time (on analytical HPLC) =
1.76 minutes.
39
Intermediate 1B
0
NH2
0,
0
3-ethyl 6-methyl 2-amino-1H-indole-3,6-dicarboxylate
[0144] In a 500 mL round-bottom flask were added methyl 4-(1-cyano-2-ethoxy-2-
oxoethyl)-3-
nitrobenzoate (14.9 g, 51.0 mmol) and zinc dust (16.7 g, 255 mmol) in acetic
acid (255 mL) to give a
gray suspension. The addition of zinc was done over 35 minutes at room
temperature over an
atmosphere of nitrogen and was fairly exothermic. The mixture was heated at
1000C for 15 hours. The
mixture was allowed to cool down, filtered through Celite TM and rinsed with
ethyl acetate. Evaporation
afforded a residue which was triturated in dichloromethane-hexanes and
afforded, after filtration 6.3 g of
the title compound. LCMS m/z 263.2 (M + H)+, retention time (on analytical
HPLC) = 1.90 minutes.
Intermediate 1C
0
/
HO
methyl 4-hydroxy-9H-pyrimido[4,5-b]indole-7-carboxylate
[0145] In a 100 mL round-bottom flask were added 3-ethyl 6-methyl 2-amino-11-1-
indole-3,6-
dicarboxylate (1.1 g, 4.19 mmol), ammonium formate (0.53 g, 8.39 mmol), and
formamide (16.7 mL,
419 mmol) to give a tan suspension which was heated to 165 C for 12 hours. The
mixture was allowed
to cool to room temperature and water was added. The resulting precipitate was
filtered, air-dried and
dried under high vacuum overnight to afford 1.1 g of the title compound. LCMS
m/z 244.2 (M + H)+,
retention time (on analytical HPLC) = 1.51 minutes.
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Intermediate 1D
0
z rµ
-N
ci
methyl 4-chloro-9H-pyrimido[4,5-b]indole-7-carboxylate
[0146] In a 100 mL round-bottom flask a mixture of methyl 4-hydroxy-9H-
pyrimido[4,5-b]indole-7-
carboxylate (1.1 g, 4.5 mmol) and phosphorous oxychloride (15 mL, 161 mmol)
was heated to 90 C for
16 hours, cooled down to room temperature and evaporated under reduced
pressure. The residue was
suspended in dichloromethane (20 mL) and filtered through Celite. Evaporation
afforded the title
compound as an orange solid (360 mg). LCMS m/z 262.0 (M+H)+, retention time
(on analytical HPLC)
= 2.02 minutes.
Example 1
0
1µ)
-N
HN
EN)
methyl 4-((3-(piperidin-1-yl)propyl)amino)-9H-pyrimido[4,5-b]indole-7-
carboxylate
[0147] In a 2-5 mL microwave vial was added methyl 4-chloro-9H-pyrimido[4,5-
b]indole-7-carboxylate
(86 mg, 0.33 mmol), triethylamine (0.09 mL, 0.66 mmol) and 3-(piperidin-1-
yl)propan-1-amine (0.078
mL, 0.49 mmol) in methanol (2 mL) and the mixture was heated to 140 C for 15
minutes in a
microwave reactor. The mixture was allowed to cool to room temperature and
evaporated under
reduced pressure. The crude material was dissolved in N,N-dimethylformamide
and purified on a
reverse-phase Zorbax SB-C18 column 21.2 x 100 mm and was eluted with Me0H -
Water - 0.1% TFA.
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Gradient: lsocratic 20% for 4 minute(s) then gradient to 100% Me0H over 15
minutes. The title
compound was obtained as the trifluoroacetic acid salt (the corresponding free
base and HCI salt were
prepared according to standard procedures known by those skilled in the art).
LCMS m/z 368.2
(M+H), retention time (on analytical HPLC) = 1.38 minutes.
Example 14
N-NNN
NI: I
,
¨N ¨N
HN HN
C) EN)
7-(1-methy1-1H-tetrazol-5-y1)-N-(3-(piperidin-1-y1)propyl)-9H-pyrimido[4,5-
b]indol-4-amine and 7-(2-
methy1-2H-tetrazol-5-y1)-N-(3-(piperidin-1-y1)propyl)-9H-pyrimido[4,5-b]indol-
4-amine.
Intermediate 14A
NC
/ N\>
¨N
HN
0
4-((3-(piperidin-1-Apropyl)amino)-9H-pyrimido[4,5-b]indole-7-carbonitrile
[0148] Starting from 4-fluoro-3-nitrobenzonitrile, Intermediate 2A was
prepared according to the
procedure described in Example I.
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Intermediate 14B
1Nz,-N
HN
/ N\S
-N
HN
N-(3-(piperidin-1-yl)propy1)-7-(2H-tetrazol-5-y1)-9H-pyrimido[4,5-b]indol-4-
amine
[0149] In a 2-5 mL microwave vial was added 44(3-(piperidin-1-yl)propyl)amino)-
9H-pyrimido[4,5-
b]indole-7-carbonitrile (47.5 mg, 0.142 mmol) and azidotributyltin (409 pl,
1.491 mmol) in
(trifluoromethyl)benzene (2 mL) to give a tan suspension. The vial was placed
in the microwave and
heated to 180 C for 30 minutes. The mixture was concentrated to dryness and
Me0H (3 mL) followed
by HCI 4M in Dioxane (1.07 mL, 4.26 mmol) was added to get a yellow solution.
To the resulting
solution was added diethyl ether (3 mL) Stirred at 20 C for 16 hours. The
solid obtained was collected
on a Buchner. The cake was washed with diethyl ether (3 x 1 mL) and with
Hexane (3 x 1 mL) and the
solid dried at 30 C under high vacuum until constant weight to afford 56 mg of
the title compound as
the HCI salt. LCMS m/z 378.2 (M+H)+, retention time (on analytical HPLC) =
1.30 minutes.
Example 14
N-NNN
NI:
-N -N
HN HN
EN)
C)
7-(1-methy1-1H-tetrazol-5-y1)-N-(3-(piperidin-1-y1)propyl)-9H-pyrimido[4,5-
b]indol-4-amine and 7-(2-
methy1-2H-tetrazol-5-y1)-N-(3-(piperidin-1-yl)propy1)-9H-pyrinnido[4,5-b]indol-
4-amine
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[0150] In a 25 mL round-bottomed flask were added N-(3-(piperidin-1-yl)propy1)-
7-(2H-tetrazol-5-y1)-
9H-pyrimido[4,5-b]indol-4-amine hydrochloride (43 mg, 0.10 mmol) and N,N-
diisopropylethylamine (36
pl, 0.21 mmol) in tetrahydrofuran (2 mL) and methanol (0.5 ml) to give a tan
suspension.
Trimethylsilyldiazomethane 2M in hexane (260 pl, 0.52 mmol) was then added.
The resulting thin
yellow suspension was stirred at 20 C for 3 hours and acetic acid (59 pl, 1.04
mmol) was added.
Stirring continuted for 30 minutes and the solvents were removed under vacuum
to afford a residue
which was purified by flash-chromatography (started with 100% dichloromethane
and gradually added a
mixture of dichloromethane : methanol : 28%wt. aqueous ammonium hydroxide (90
: 10 : 1) increments
to complete with 100% dichloromethane : methanol : 28%wt, aqueous ammonium
hydroxide (90 : 10 :
1). The first eluting product obtained was of 7-(2-methyl-2H-tetrazol-5-y1)-N-
(3-(piperidin-1-yl)propy1)-
9H-pyrimido[4,5-b]indol-4-amine (19 mg). LCMS m/z 392.2 (M+H)+, retention time
(on analytical
HPLC) = 1.44 minute. The second eluting product was of 7-(1-methyl-1H-tetrazol-
5-y1)-N-(3-(piperidin-
1-yl)propy1)-9H-pyrimido[4,5-13]indol-4-amine (5 mg). LCMS m/z 392.2 (M+H)+,
retention time (on
analytical HPLC) = 1.27 minutes.
Example 15
0 1- NaH, DMF 0
Me0 NO2 NC-CH2CON me,0 Me0Na, Me0H
/ NH2
F FeC13 , H20 l'yCO2Me
Zn, DMF, water
0NH2
Intermediate 15A Intermediate 15 B
0
0
Me'0 1- POCI3, 175 C, 15 min Me,o
, N N
¨
2- Et3N, Me0H
HN
¨ H2
Intermediate 15C Example 15 \--"\¨NO
[0151] NaH (3.41 g, 85 mmol) was added portionwise to a cold solufion of 2-
cyanoacetamide (7.18 g,
85 mmol) in DMF (53 mL). After 30 minutes at room temperature, a solution of
methyl 4-fluoro-3-
nitrobenzoate (8.5 g, 42.7 mmol) in 15 mL of DMF was added dropwise. After 3
hours a mixture of ice,
water and 12 mL HCI (10%) were added. The resulting solid was filtered, rinsed
with water and dried
under high vacuum overnight to give 9.1 g of methyl 4-(2-amino-1-cyano-2-
oxoethyl)-3-nitrobenzoate:
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1H NMR (400 MHz, DMSO-d6) 6 ppm 3.93 (s, 3 H) 5.78 (s, 1 H) 7.77 (s, 1 H) 7.91
(d, J=7.83 Hz, 1 H)
8.04 (s, 1 H) 8.39 (dd, J=8.02, 1.76 Hz, 1H) 8.56 (d, J=1.56 Hz, 1 H).
[0152] Ferric chloride hexahydrate (1.540 g, 5.70 mmol) and zinc (1.242 g,
19.00 mmol) were added
to a solution of the crude cyano-amide prepared above (0.5 g, 1.900 mmol) in
DMF (4.75 mL) and
water (4.75 mL) to give a yellow suspension. After the exotherm, the mixture
was heated to 100 C for
45 minutes and then slowly cooled to 20 C and stirred for 22 hours. The solid
was filtered, washed
with DMF (3 x 3 mL) and the filtrate was diluted with water (40 mL) while
stirring at 0 C. The solid was
filtered and the cake washed with water (2 x 5 mL). The solid contains mostly
impurities. The aqueous
layer was extracted with Et0Ac (3 x 50 mL) and the combined organic layers
were washed with water
(50 mL) and then with brine (30 mL). The organic layer was dried over anh.
MgSO4, filtered and
concentrated to give 287 mg as a brown solid which was treated with acetone (6
mL) to give a solid
suspension which was diluted with hexane (5 mL). Then the solid was collected
and dried at 40 C
under high vacuum until constant weight to give Intermediate 15B methyl 2-
amino-3-carbamoy1-1H-
indole-6-carboxylate (162 mg, 36.6% yield) as an off-white solid: 1H NMR (400
MHz, DMSO-d6) 6 ppm
3.80 (s, 3 H) 6.62 (br. s., 2 H) 7.04 - 7.18 (m, 2 H) 7.53 - 7.63 (m, 2 H)
7.72 (s, 1 H) 10.80 (s, 1 H); MS
m/z 232.2 (M+H)+; HPLC ca. 96%, RT = 1.37 minutes.
[0153] A mixture of Intermediate 15B (0.100 g, 0.429 mmol), methyl 2-(pyridin-
3-yl)acetate (0.130 g,
0.858 mmol) and sodium nnethoxide 25%wt in Me0H (0.196 mL) in methanol (0.954
mL) was placed in
the microwave oven and heated to 140 C for 45 minutes. After cooling, AcOH
(0.050 mL, 0.879 mmol)
was added and the resulting slurry was stirred at 20 C for 1 hour. The solids
were filtered, washed with
Me0H (3 x 0.5 mL),dried at 20 C under high vacuum until constant weight to
give Intermediate 15C:
methyl 4-hydroxy-2-(pyridin-3-yInnethyl)-9H-pyrimido[4,5-13]indole-7-
carboxylate (82 mg, 57.2% yield) as
a brown solid: 1H NMR (400 MHz, DMSO-d6) 6 ppm 3.87 (s, 3 H) 4.09 (s, 2 H)
7.34 - 7.40 (m, 1 H) 7.79
(dt, J=8.1, 1.8 Hz, 1 H) 7.83 (dd, J=8.2, 1.2 Hz, 1 H) 7.99 (d, J=0.8 Hz, 1 H)
8.02 (d, J=8.2 Hz, 1 H)
8.48 (dd, J=4.9, 1.4 Hz, 1 H) 8.60 (d, J=2.0 Hz, 1 H) 12.49 (br. s., 2 H): MS
m/z 335.2 (M+H)+; HPLC
95.2% @ 220 nm and 92.8% @ 254 nm, RT = 1.42 minutes.
[0154] A mixture of methyl 4-hydroxy-2-(pyridin-3-ylmethyl)-9H-pyrimido[4,5-
b]indole-7-carboxylate
(0.050 g, 0.150 mmol) in POCI3 (0.948 mL, 10.17 mmol) was placed in a vial and
heated to 175 C for
15 minutes in a microwave oven. After cooling, the reaction mixture was poured
into ice-water (19 mL)
then basified to pH 8 by the slow addition of 50% aq. NaOH (2.7 mL) and
finally diluted with Et0Ac
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(20 mL), the solids were filtered (first crop of chloride) and the aqueous
layer was extracted with Et0Ac
(20 mL) and the combined organic layers were dried over anh. MgSO4, filtered
and concentrated to
dryness to afford an additional 35 mg crop of the desired chloride derivative.
The combined isolated
crops of methyl 4-chloro-2-(pyridin-3-ylmethyl)-9H-pyrimido[4,5-b]indole-7-
carboxylate (53 mg, 100%
yield) were used directly in the next step.
[0155] A mixture of methyl 4-chloro-2-(pyridin-3-ylmethyl)-9H-pyrimido[4,5-
b]indole-7-carboxylate
(0.053 g, 0.150 mmol), 3-(piperidin-1-yl)propan-1-amine (0.072 mL, 0.451 mmol)
and triethylamine
(0.063 mL, 0.451 mmol) in Me0H (2.5 mL) was placed in a vial and heated to 140
C for 15 minutes in a
microwave oven for 15 minutes. After cooling and evaporation of the solvent,
the residue was purified
by flash chromatography to give 23 mg of a yellow oil + solid which was
diluted with CH3CN (3 mL) and
stirred for 30 minutes. The solids were filtered and washed with CH3CN (2 x
0.5 mL), then dried at
30 C under high vacuum until constant weight to afford the compound of Example
15: methyl 4-((3-
(piperidin-1-yl)propyl)amino)-2-(pyridin-3-ylmethyl)-9H-pyrimido[4,5-b]indole-
7-carboxylate (11 mg, 16%
yield) as a tan solid: 1H NMR (400 MHz, DMSO-d6) 5 ppm 1.31 - 1.44 (m, 2 H)
1.44 - 1.56 (m, 4 H)
1.71 - 1.86 (m, 2 H) 2.17 - 2.47 (m, 6 H) 3.56 - 3.66 (m, 2 H) 3.88 (s, 3 H)
4.08 (5, 2 H) 7.28 - 7.34 (m, 1
H) 7.43 (t, J=5.5 Hz, 1 H) 7.76 (dt, J=7.8, 2.0 Hz, 1 H) 7.81 (dd, J=8.2, 1.4
Hz, 1 H) 7.99 (d, J=1.4 Hz, 1
H) 8.35 (d, J=8.2 Hz, 1 H) 8.41 (dd, J=4.7, 1.6 Hz, 1 H) 8.59 (d, J=2.0 Hz, 1
H) 12.08 (s, 1 H); MS m/z
459.2 (M+H)+; HPLC >99.5%, RT = 1.43 minutes.
Example 22
0
Me
Me"0
N 411 N 11.
1" Et3N, Me0H /
/
0-Bn
CI HN 0-E1
2- H2 10% Pd/C
NH4HCO2, Me0H Example 22
[0156] A mixture of methyl 2-((benzyloxy)(phenyl)methyl)-4-chloro-9H-
pyrimido[4,5-b]indole-7-
carboxylate (prepared as described for Example 15, 0.228 g, 0.498 mmol), 3-
(piperidin-1-yl)propan-1-
amine (0.158 mL, 0.996 mmol) and triethylamine (0.173 mL, 1.245 mmol) in Me0H
(3.8 mL) was
placed in the microwave oven and heated to 140 C for 30 minutes. After cooling
to room temperature,
the mixture was concentrated to dryness and the residue was purified by flash
chromatography to
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afford methyl 2-
((benzyloxy)(phenyl)methyl)-4-((3-(piperidin-1-yl)propyl)amino)-9H-
pyrimido[4,5-
b]indole-7-carboxylate (172 mg, 61.3% yield) as a light yellow solid: 1H NMR
(400 MHz, DMSO-d6) 6
ppm 1.29 - 1.42 (m, 2 H) 1.42 -1.56 (m, 4 H) 1.72 - 1.91 (m, 2 H) 2.18 - 2.47
(m, 6 H) 3.66 (tt, J=13.2,
6.6 Hz, 2 H) 3.88 (s, 3 H) 4.54 (d, J=11.7 Hz, 1 H) 4.64 (d, J=12.1 Hz, 1 H)
5.50 (s, 1 H) 7.21 -7.43 (m,
8 H) 7.51 (t, J=5.9 Hz, 1 H) 7.57 (d, J=7.0 Hz, 2 H) 7.82 (dd, J=8.2, 1.2 Hz,
1 H) 8.00 (d, J=1.2 Hz, 1 H)
8.38 (d, J=8.2 Hz, 1 H) 12.22 (s, 1 H); HRMS miz 564.2979 (M+H)+; HPLC 99.6%,
RT = 2.02 minutes.
[0157] Hydrogenolysis of the benzyl group was performed on derivative methyl 2-
(
0042 g, 0.075 mmol) in the presence of Pd-C 10%wt. (50% wet) (0.159 g, 0.075
mmol) and
ammonium formate (0.235 g, 3.73 mmol) under hydrogen in methanol. After 26
hours of stirring at
55 C, the reaction mixture was filtered over Celite, rinsed with Me0H, and
concentrated to dryness on
a rotovap to give 133 mg of a residue which was purified by RP HPLC using a
Zorbax SB-C18 column
21.2 x 150 mm eluted with Me0H - water - 0.1% TFA to afford 21.9 mg (50%
yield) of Example 22 :
methyl 2-
(hydroxy(phenyl)methyl)-4-((3-(piperidin-1-y1)propyl)amino)-9H-pyrimido[4,5-
b]indole-7-
carboxylate, TFA salt as a white solid: 1H NMR (400 MHz, DMSO-d6) 6 ppm 1.30 -
1.43 (m, 1 H) 1.52 -
1.73 (m, 3 H) 1.79 (br. d, J=14.5 Hz, 2 H) 1.96- 2.09 (m, 2 H) 2.54 (s, 1 H)
2.74 -2.89 (m, 2 H) 3.11 (dt,
J=10.4, 5.4 Hz, 2 H) 3.38 (d, J=12.1 Hz, 2 H) 3.71 -3.77 (m, 2 H) 3.88 (s, 3
H) 5.63 (s, 1 H) 7.19 - 7.26
(m, 1 H) 7.27 - 7.35 (m, 2 H) 7.48 - 7.56 (m, 2 H) 7.62 (t, J=5.9 Hz, 1 H)
7.85 (dd, J=8.2, 1.4 Hz, 1 H)
8.03 (d, J=1.4 Hz, 1 H) 8.38 (d, J=8.2 Hz, 1 H) 8.92 (br. s., 1 H) 12.21 (s, 1
H); HRMS nn/z 474.2511
(M+H); HPLC >99%, RI = 1.68 minutes.
[0158] Dess-Martin periodinane reagent (22.67 mg, 0.053 mmol) was added to a
mixture of methyl 2-
(hydroxy(phenyl)rnethyl)-4-((3-(pi peridin-1-yl)propyl)arnino)-9H-
pyrirnido[4,5-b]indole-7-carboxylate
(compound of Example 22) and TFA (15.7 mg, 0.027 mmol) in DCM (1000 pL, 15.54
mmol) to give a
light orange solution. After 1 h, the solvent was evaporated and the residue
was purified by flash
chromatography to afford Example 23: methyl 2-benzoy1-44(3-(piperidin-1-
yl)propyl)amino)-9H-
pyrimido[4,5-b]indole-7-carboxylate (10 mg, 79% yield) as a bright yellow
solid: 1H NMR (400 MHz,
DMSO-d6) 6 ppm 1.27- 1.36 (m, 2 H) 1.36- 1.48 (m, 4 H) 1.74- 1.88 (m, 2 H)
2.14- 2.44 (m, 6 H) 3.52
- 3.68 (m, 2 H) 3.91 (s, 3 H) 7.54 (t, J=7.8 Hz, 2 H) 7.69 (t, J=7.4 Hz, 1 H)
7.76 (t, J=5.1 Hz, 1 H) 7.90
(dd, J=8.2, 1.4 Hz, 1 H) 7.94 (d, J=7.0 Hz, 2 H) 8.10 (d, J=1.4 Hz, 1 H) 8.52
(d, J=8.2 Hz, 1 H) 12.43 (s,
1 H) ; HRMS rrilz 472.2342 (M+H)4; HPLC 97.1% @ 220 nm and 98.9% @ 254 nm, RI
= 1.86
minutes.
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Example 25
0 0 0 H
Me'0 H N Me'0AJ Me'0 N
/ rs V NH201-I HC1 rs 1- NH3, DCM F.4%
-N PyridineTaO -NN
HN = 0 meoH
=/CF 2-12, Et3N, DCM HN -
N
=
Cõ
?- CF3
2- MCI, DMAP,
ND
Et3N, DCM NO NO
Example 25
Example 33 Intermediate 33A
[0159] Hydroxylamine hydrochloride (0.08 g, 1.2 mmol) was added to methyl 4-
((3-(piperidin-1-
yl)propyl)amino)-2-(3-(2,2,2-trifluoroacetyl)benzy1)-9H-pyrimido[4,5-b]indole-
7-carboxylate (compound
of Example 33) (0.285 g, 0.515 mmol) in Me0H (4.00 mL) and pyridine (0.666 mL)
to give a yellow
solution. After heating at 60 C 5 days, the mixture was concentrated to
dryness and the residue was
dissolved into DCM (75 mL) and Me0H (15 mL) and this solution was washed with
sat. NaHCO3 (20
mL), The aqueous layer was extracted two time with a mixture of CH2Cl2 (50 mL)
and Me0H (10 mL)
and the combined organic layers were dried over anh. MgSO4, filtered and
concentrated to dryness to
give methyl 44(3-(piperidin-111)propyl)amino)-2-(3-(2,2,2-trifluoro-1-
(hydroxyimino)ethyl)benzy1)-9H-
pyrinnido[4,5-b]indole-7-carboxylate (293 mg, 100% yield) as an off-white
solid: 1H NMR (400 MHz,
DMSO-d6) 5 ppm 1.32 - 1.43 (m, 2 H) 1.44 - 1.56 (m, 4 H) 1.76 - 1.87 (m, 2 H)
2.23 - 2.44 (m, 6 H) 3.57
- 3.67 (m, 2 H) 3.88 (s, 3 H) 4.04 - 4.12 (m, 2 H) 7.25 - 7.33 (m, 1 H) 7.34 -
7.46 (m, 2 H) 7.50 (m,
J=7.6, 4.1 Hz, 1 H) 7.55 (d, J=7.4 Hz, 1 H) 7.81 (dd, J=8.2, 1.2 Hz, 1 H) 7.99
(d, J=1.2 Hz, 1 H) 8.36 (d,
J=8.2 Hz, 1 H) 12.07 (d, J=3.5 Hz, 1 H); MS nn/z 569.2 (M+H)+; HPLC >95%, RI =
1.88 minutes.
[0160] Tosyl chloride (0.048 g, 0.251 mmol) was added portionwise to a cold
mixture of methyl 4-((3-
(piperidin-111)propyl)amino)-2-(3-(2,2,2-trifluoro-1-
(hydroxyimino)ethyl)benzyl)-9H-pyrimido[4,5-
b]indole-7-carboxylate (0.130 g, 0.229 mmol), 4-dinnethylaminopyridine (2.79
mg, 0.023 mmol) and
triethylamine (0.038 mL, 0.274 mmol) in DCM (10.00 mL) to give a white
suspension. After one hour at
room temperature, the amber solution was diluted with DCM (10 mL) and washed
with water (3 x 10
mL). The organic layer was dried over anh. MgSO4, filtered and concentrated to
dryness to give methyl
4-((3-(piperidin-1-yhpropyhamino)-2-(3-(2,2,2-trifluoro-1-
((tosyloxy)imino)ethyl)benzy1)-9H-pyrimido[4, 5-
b]indole-7-carboxylate (188 mg, 99% yield) as a tan foam: MS m/z 723.2 (M+H)+;
HPLC >89%, RI =
2.09 minutes.
[0161] To a solution of methyl
4-((3-(piperidin-1-yl)propyl)amino)-2-(3-(2,2,2-trifluoro-1-
((tosyloxy)imino)ethyl)benzy1)-9H-pyrimido[4,5-b]indole-7-carboxylate (0.188
g, 0.260 mmol) in DCM
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(5.00 mL) cooled to -78 C, ammonia (1.689 mL, 78 mmol) was added and the tube
was sealed and
warmed to 20 C. The reaction mixture turned blue over time and after 3.5 hours
it was cooled again to
-78 C and then slowly warmed to 20 C with septa + nitrogen outlet to evaporate
most of the ammonia.
After 3.5 hours, the reaction mixture was filtered over Buchner to remove most
of the ammonium p-
toluenesulfonate), the solids were washed with DCM (3 x 1.5 mL) and the
filtrate was concentrated to
dryness to give a yellow foam which was purified by flash chromatography to
afford methyl 4-((3-
(piperidin-1-yl)propyl)amino)-2-(3-(3-(trifluoromethyl)diaziridin-3-yl)benzy1)-
9H-pyrimido[4,5-b]indole-7-
carboxylate (115 mg, 78% yield) as a white foam: 1H NMR (400 MHz, DMSO-dÃ) 6
ppm 1.31 - 1.43 (m,
2 H) 1.50 (quin, J=5.3 Hz, 4 H) 1.80 (dt, J=14.1, 7.0 Hz, 2 H) 2.32 (m, J=6.7,
6.7 Hz, 6 H) 3.58 - 3.67
(m, 2 H) 3.88 (s, 3 H) 3.93 (br. d, J=7.4 Hz, 1 H) 4.05 (br. d, J=8.6 Hz, 1 H)
4.07 (s, 2 H) 7.32 - 7.43 (m,
3 H) 7.47 (m, J=6.3 Hz, 1 H) 7.59 (s, 1 H) 7.81 (dd, J=8.4, 1.2 Hz, 1 H) 7.99
(d, J=1.2 Hz, 1 H) 8.35 (d,
J=8.4 Hz, 1 H) 12.07 (s, 1 H); MS m/z 568.2 (M+H)r; HPLC >94%, RT = 1.72
minutes.
[0162] Iodine (0.028 g, 0.111 mmol) was added to a mixture of methyl 4-((3-
(piperidin-1-
y0propyl)amino)-2-(3-(3-(trifluoromethyl)diaziridin-311)benzyl)-9H-
pyrimido[4,5-blindole-7-carboxylate
(0.060 g, 0.106 mmol) and triethylamine (0.044 mL, 0.317 mmol) in DCM (2 mL)
to give a yellow
solution. After 15 minutes, the solvent was evaporated under reduced pressure
to give a residue which
was purified by flash chromatography to afford 95 mg as a yellow foam. The
foam was dissolved in
DCM (15 mL) and washed with sat. NaHCO3 (10 mL). The organic layer was dried
over anh. MgSO4,
filtered and concentrated to dryness to afford the compound of Example 25:
methyl 4-((3-(piperidin-1-
yl)propyl)amino)-2-(3-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzy1)-9H-
pyrimido[4,5-13]indole-7-
carboxylate (50 mg, 84% yield) as a light yellow solid: 1H NMR (400MHz, DMSO-
d6) 6 ppm 1.36 (m,
J=5.1 Hz, 2 H) 1.48 (quin, J=5.5 Hz, 4 H) 1.76 (quin, J=7.0 Hz, 2 H) 2.30 (br.
t, J=6.5, 6.5 Hz, 6 H) 3.54
- 3.67 (m, 2 H) 3.88 (s, 3 H) 4.09 (s, 2 H) 7.14 (d, J=7.8 Hz, 1 H) 7.26 (s, 1
H) 7.38 - 7.47 (m, 2 H) 7.52
(d, J=7.4 Hz, 1 H) 7.81 (d, J=8.2 Hz, 1 H) 7.99 (s, 1 H) 8.35 (d, J=8.2 Hz, 1
H) 12.06 (s, 1 H); HRMS
m/z 566.2497 (M+H)+; HPLC 94.5% @ 220 nm and 92.9% @ 254 nm, RT = 2.05
minutes.
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Examples 33 and 34
0 H 0
MeND N = Br 0
FM,
N
/ 1- CO, Et3SIH Me-T2 N OH De. martin 0
/ rs 11-1-7 CF2
"TN / rµ\I c,3 Parlodlnane
HN
2- Me2SICF3, CsF T-N CH2Cl2
-ND HN HN TFA
-ND
\-ND Example 33
Example 28 Example 34
[0163] CO was bubbled into a solution of methyl 2-(3-bromobenzyI)-4-((3-
(piperidin-1-
yl)propyl)amino)-9H-pyrimido[4,5-b]indole-7-carboxylate (prepared as for
Example 15, 0.090 g, 0.168
mmol), triethylsilane (0.054 mL, 0.336 mmol) and PdC12 (dppf) (6.14 mg, 8.39
pmol) and the mixture
was heated overnight at 95 C. The crude mixture was purified by preparative
HPLC to give 44mg of
carbonylated product as a solid: 1H NMR (400 MHz, DMSO-d6) 6 ppm 1.28 - 1.40
(m, 1 H) 1.50 - 1.72
(m, 3 H) 1.73 - 1.84 (m, 2 H) 1.95 -2.06 (m, 2 H) 2.74 - 2.87 (m, 2 H) 3.03 -
3.12 (m, 2 H) 3.33 - 3.41
(m, 2 H) 3.88 (s, 3 H) 4.20 (s, 2 H) 7.46 - 7.60 (m, 2 H) 7.73 (d, J=7.83 Hz,
1 H) 7.79 (d, J=7.43 Hz, 1
H) 7.84 (dd, J=8.22, 1.57 Hz, 1 H) 7.91 (s, 1 H) 8.01 (d, J=1.17 Hz, 1 H) 8.37
(d, J=8.61 Hz, 1 H) 8.92
(br. s., 1 H) 10.00 (s, 1 H) 12.16 (s, 1 H).
[0164] Trimethyl(trifluoromethyl)silane (0.7 mL, 3.5 mmol) was added to a
mixture of methyl 2-(3-
formylbenzy1)-44(3-(piperidin-111)propyl)amino)-9H-pyrimido[4,5-b]indole-7-
carboxylate (0.260 g, 0.535
mmol) and cesium fluoride (5.69 mg, 0.037 mmol) cooled to 0 C. After stirring
for 2 days at room
temperature, HCI conc. (0.5 mL) in 2 mL of water was added and stirred 15 min.
The mixture was
diluted with ethyl acetate, neutralized with solid Na2CO3, the phases were
separated and the aqueous
layer was extracted 2 times with EA. The combined organic layers were washed
with water, dried over
anh. MgSO4, filtered and the solvent removed to give a residue which was
purified by preparative
HPLC to give 126 mg of corresponding TFA salt: 1H NMR (400 MHz, DMSO-d6) 5 ppm
1.24 - 1.43 (m,
1 H) 1.49 - 1.73 (m, 4 H) 1.73 - 1.82 (m, 2 H) 1.97 - 2.07 (m, 2 H) 2.80 (q,
J=11.70 Hz, 2 H) 3.02 - 3.12
(m, 2 H) 3.36 - 3.42 (m, 2 H) 3.88 (s, 3 H) 4.10 (s, 2 H) 5.12 (q, J=7.17 Hz,
1 H) 6.81 (br. s., 1 H) 7.30 -
7.35 (m, 2 H) 7.36 - 7.42 (m, 1 H) 7.48 - 7.58 (m, 2 H) 7.84 (dd, J=8.41, 1.37
Hz, 1 H) 8.01 (s, 1 H) 8.37
(d, J=8.61 Hz, 1 H) 8.95 (br. s., 1 H) 12.17 (s, 1 H); MS m/z 554.2 (M+H)+;
HPLC RI 2.142 minutes.
[0165] Dess-Martin periodinane (56.5 mg, 0.133 mmol) was added to methyl 4-((3-
(piperidin-1-
yl)propyl)amino)-2-(3-(2,2,2-trifluoro-1-hydroxyethyl)benzy1)-9H-pyrimido[4,5-
13]indole-7-carboxylate (20
mg, 0.036 mmol) in DCM (753 pL) to give a white suspension. After stirring at
20 C for 1 hour the
mixture was purified by flash chromatography to give methyl 4-((3-(piperidin-1-
yl)propyl)amino)-2-(3-
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(2,2,2-trifluoroacetyl)benzyI)-9H-pyrimido[4,5-b]indole-7-carboxylate as
Example 33 (18.4 mg, 92%
yield) as a yellow solid: 1H NMR in DMSO-d6 was consistent with the desired
product but complicated
because of the presence of hydrate forms; HRMS rniz 554.2384 (M+H)T; HPLC
>95%, RT = 1.76 and
1.87 minutes (ketone + hydrate).
Example 35
Nz=N
N
¨N
HN
Intermediate 35B: N2 isomer precursor
IMSN N-,N K2c03 N¨N
3
NC Mel I
Bu2SnO
10/ +
F Toluene N CH3CN
Intermediate 35 A N2 isomer Ni isomer
Intermediate 35 B
[0166] A mixture of 4-fluorobenzonitrile (5 g, 41.3 mmol), dibutyltin oxide
(2.055 g, 8.26 mmol), and
trimethylsilyl azide (8.22 mL, 61.9 mmol) in toluene (165 mL) was heated to
100 C and stirred for 16.5
hours. After cooling to room temperature, the organic layer was extracted with
NaOH 1M (83 mL) and
the aqueous layer was washed with Et0Ac (2 x 85 mL). The aqueous layer was
acidified with HCI 2M
(41.3 mL) to pH 2. The aqueous mixture was extracted twice with Et0Ac (200 mL
then 100 mL) and
the combined organic layers were washed with brine (60 mL), dried over anh.
MgSO4, filtered and
concentrated to dryness to afford Intermediate 35A (5-(4-fluorophenyI)-2H-
tetrazole, 6.61 g, 98% yield)
as a white solid: 1H NMR (400 MHz, DMSO-d6) 6 ppm 7.42 - 7.53 (m, 2 H) 8.04 -
8.14 (m, 2 H); MS m/z
165.2 (M+H); HPLC >99.5%, RT = 1.96 minutes.
[0167] A mixture of 5-(4-fluorophenyI)-2H-tetrazole (6.61 g, 40.3 mmol), K2CO3
(6.68 g, 48.3 mmol),
and iodomethane (3.02 mL, 48.3 mmol) in acetonitrile (115 mL) was heated to
reflux (ca. 82 C) for one
hour. After cooling, the mixture was concentrated to dryness and the residue
was partitioned between
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water (75 mL) and Et0Ac (100 mL). The layers were separated, the aqueous layer
was back-extracted
with Et0Ac (50 mL) and the combined organic layers were washed with water (50
mL) and brine (50
mL). The organic layer was dried over anh. MgSO4, filtered and concentrated to
give 9.5 g as a
colorless oil that solidified upon standing. The residue was purified by flash
chromatography to give 2
main products: Intermediate 35B as the N2 isomer: 5-(4-fluorophenyI)-2-methyl-
2H-tetrazole (5.09 g,
70.9% yield) as a white solid: No NOE observed between the methyl group at
4.42 ppm and the
aromatic protons; 1H NMR (400 MHz, DMSO-d6) 6 ppm 4.42 (s, 3 H) 7.33 - 7.45
(m, 2 H) 8.03 - 8.14
(m, 2 H); MS m/z 179.2 (M+H)+; HPLC >99.5%, RT = 1.75 minutes.
[0168] The N1 isomer: 5-(4-fluorophenyI)-1-methyl-1H-tetrazole (1.87 g, 26.1%
yield) as a white solid:
the NOE observed between the methyl group at 4.16 ppm and the two aromatic
protons at 7.89 - 7.97
ppm confirms the structure; 1H NMR (400 MHz, DMSO-d6) 6 ppm 4.16 (s, 3 H) 7.43-
7.53 (m, 2 H) 7.89
- 7.97 (m, 2 H); MS m/z 179.2 (M+H)+; HPLC >99.5%, RT = 1.29 minutes.
Intermediates 35 C et D
Me-N, = 1- HNO3, H2SO4 me--N, NO
2 zFen013 6H20
N
F 2- NC-CH2CONH2
CN
DMF
/ NH2
NaH, THE 0 NH2 Water
0 NH2
Intermediate 35B Intermediate 35C Intermediate 35 D
[0169] A solution of Intermediate 35B (5-(4-fluorophenyI)-2-methyl-2H-
tetrazole, 1 g, 5.61 mmol) in
sulfuric acid (16.45 mL, 309 mmol) was cooled to 0 C and then fuming nitric
acid (0.288 mL, 6.17
mmol) was added dropwise. After 2.5 hours, more fumic nitric acid was added
(0.065 mL, 1.403 mmol)
was added and the mixture allowed to warm to 20 C. After 5 hours, the mixture
was poured into a 2:1
ice-water mixture (150 mL) leading to the formation of a white suspension.
After 30 minutes, the solid
was filtered, washed with water (4 x 10 mL, until neutral pH of the washes),
dried at 25 C under high
vacuum until constant weight: 5-(4-fluoro-3-nitrophenyI)-2-methyl-2H-tetrazole
(1.16 g, 93% yield) as an
off-white solid: 1H NMR (400 MHz, DMSO-d6) 6 ppm 4.47 (s, 3 H) 7.81 (dd,
J=11.2, 8.8 Hz, 1 H) 8.44
(ddd, J=8.7, 4.2, 2.3 Hz, 1 H) 8.68 (dd, J=7.2, 2.2 Hz, 1 H); MS m/z 224.2
(M+H)+; HPLC 98.3%, RT =
1.72 minutes.
[0170] A solution of 2-cyanoacetamide (0.888 g, 10.56 mmol) in DMF (2.268 mL)
was added to a
suspension of sodium hydride 60%wt. in mineral oil (0.443 g, 11.08 mmol) in
DMF (5.67 mL) to give a
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grey suspension. After cooling to 0 C (Note: Hydrogen Gas evolution) the
resulting mixture was stirred
at 0 C for 30 minutes. Then a solution of 5-(4-fluoro-3-nitropheny1)-2-methyl-
2H-tetrazole (1.15 g, 5.15
mmol) in DMF (2.3 mL) was added to give a deep purple solution. After 3 hours,
the reaction mixture
was slowly poured into an ice - water mixture (33.0 mL) and conc.HCI (0.952
mL). The resulting yellow
slurry was stirred for 30 minutes, the solid was filtered, washed with water
(3 x 5 mL) and then with
Hexane (2 x 5 mL), dried at 40 C under high vacuum until constant weight to
give 2-cyano-2-(4-(2-
methy1-2H-tetrazol-5-y1)-2-nitrophenyl)acetamide (1.41 g, 95% yield) as a
yellow solid: 1H NMR (400
MHz, DMSO-d6) 6 ppm 4.49 (s, 3 H) 5.77 (s, 1 H) 7.77 (s, 1 H) 7.95 (d, J=8.2
Hz, 1 H) 8.03 (s, 1 H)
8.51 (dd, J=8.2, 1.8 Hz, 1 H) 8.70 (d, J=1.8 Hz, 1 H); MS miz 288.1 (M+H)+;
HPLC 96.4% @ 220 nm,
RT = 1.31 minutes.
[0171] Ferric chloride hexahydrate (2.82 g, 10.44 mmol) and zinc (2.276 g,
34.8 mmol) were added
portionwise to a mixture of 2-cyano-2-(4-(2-methy1-2H-tetrazol-5-y1)-2-
nitrophenyl)acetamide (1 g, 3.48
mmol) in DMF (8.71 mL) and water (8.71 mL) to give a yellow suspension which
was heated to 100 C
for 1.25 hour. The mixture was then cooled to 20 C, diluted with Me0H (50.0
mL), filtered over Celite
and concentrated under reduced pressure to ca. 20 mL (to remove most of the
Me0H). Then the
mixture was diluted with water (50 mL) and Et0Ac (100 mL), stirred vigorously
and filtered. The
aqueous layer was extracted with Et0Ac (2 x 50 mL) and the combined organic
layers were washed
with sat. NaHCO3 (50 mL) and brine (50 mL). The organic layer was dried over
anh. MgSO4, filtered
and concentrated to give 489 mg as a purple solid which was purified by flash
chromatography to give
2-amino-6-(2-methy1-2H-tetrazol-5-y1)-1H-indole-3-carboxamide (356 mg, 39.7%
yield) as a purple
solid: 1H NMR (400 MHz, DMSO-d6) 6 ppm 4.38 (s, 3 H) 6.57 (s, 2 H) 7.01 (s, 2
H) 7.61 - 7.69 (m, 2 H)
7.81 (s, 1 H) 10.77 (s, 1 H); MS miz 258.2 (M+H)+; HPLC ca. 78%, RT = 1.34
minutes.
Me-4,
1- Ph-CH2-0O2Me
/ NH2 Me0Na, Me0H
N
NH2 2- POCI3, 175 C
\
0
3- Et3N, Me0H, 140 C HN\ __ TFA
Intermediate 35D
H2N \-N1/ )
"-NI/ Example 35
[0172] A mixture of Intermediate 35D (2-amino-6-(2-methy1-2H-tetrazol-5-y1)-1H-
indole-3-
carboxamide, 0.35 g, 1.361 mmol), methyl 2-phenylacetate (0.288 mL, 2.041
mmol) and sodium
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methoxide 25%wt. in Me0H (0.467 mL) and methanol (3.03 mL) in a microwave tube
was placed in the
microwave oven and heated to 140 C for one hour. After cooling to room
temperature and dilution with
water (1 mL) and AcOH (4 mL) the mixture was stirred for 30 minutes to allow
crystallization. The solid
was filtered, washed with Me0H (5 x 1 mL), dried at 40 C under high vacuum
unfil constant weight to
afford 2-benzy1-7-(2-methyl-2H-tetrazol-5-y1)-9H-pyrimido[4,5-b]indol-4-ol
(220 mg, 45.2% yield) as a
brown solid. 1H NMR (400 MHz, DMSO-d6) 6 ppm 4.03 (5, 2 H) 4.43 (s, 3 H) 7.24 -
7.29 (m, 1 H) 7.34
(t, J=7.8 Hz, 2 H) 7.37 - 7.43 (m, 2 H) 7.92 (dd, J=8.0, 1.4 Hz, 1 H) 8.04 -
8.10 (m, 2 H) 12.38 (s, 1 H)
12.47(s, 1 H); MS m/z 358.2 (M+H)+; HPLC 82.9%, RT = 1.89 minutes.
[0173] In a 2-5 mL microwave vial was added the crude product 2-benzy1-7-(2-
methyl-2H-tetrazol-5-
y1)-9H-pyrimido[4,5-b]indol-4-ol (0.220 g, 0.616 mmol) and POCI3 (3.90 mL,
41.9 mmol) to give a brown
suspension. The vial was placed in the microwave oven and heated to 175 C for
15 min., then allowed
to cool. The reaction mixture was then poured into water and ice mixture (80
ml), basified to pH 8 by
slow addition of NaOH 50%wt (11 mL) and then Et0Ac (80 mL). Some solids were
filtered and the
layers separated. The aqueous layer was extracted with Et0Ac (80 mL) and the
organic layer was dried
over anh. MgSO4, filtered and concentrated to dryness to give the
corresponding chloro derivative: 2-
benzy1-4-chloro-7-(2-methyl-2H-tetrazol-5-y1)-9H-pyrimido[4,5-b]indole (189
mg, 82% yield) as a brown
solid 1H NMR (400 MHz, DMSO-d6) 6 ppm 4.31 (s, 2 H) 4.46 (5, 3 H) 7.20 - 7.26
(m, 1 H) 7.28 - 7.39
(m, 4 H) 8.09 (dd, J=8.2, 1.2 Hz, 1 H) 8.21 - 8.25 (m, 1 H) 8.39 (d, J=8.2 Hz,
1 H) 12.93 (s, 1 H); MS
nn/z 376.2 (M+H)+; HPLC 95.6%, RI = 2.30 minutes.
[0174] A mixture of 2-benzy1-4-chloro-7-(2-methyl-2H-tetrazol-5-y1)-9H-
pyrimido[4,5-b]indole prepared
as described above (0.050 mg, 0.133 mmol) and Et3N (0.037 mL, 0.266 mmol) and
3-(piperidin-1-
yl)propan-1-amine (0.033 mL, 0.200 nnmol) in Me0H (0.6 nnL) was heated 25
minutes at 140 C in a
microwave oven. After cooling and evaporation of the solvent, the residue was
purified by RP-HPLC
(Me0H-water (0.5% TEA) 20% to 100% Me0H to afford 55 mg of Example 35: 2-
benzy1-7-(2-methyl-
2H-tetrazol-5-y1)-N-(3-(piperidin-1-yl)propy1)-9H-pyrimido[4,5-b]indol-4-amine
2,2,2-trifluoroacetate; 1H
NMR (400 MHz, DMSO-d6) 6 ppm 1.27- 1.41 (m, 1 H) 1.53- 1.72 (m, 3 H) 1.79 (d,
J=13.69 Hz, 2 H)
1.98 - 2.09 (m, 2 H) 2.75 -2.87 (m, 2 H) 3.09 (dt, J=10.27, 5.23 Hz, 2 H) 3.39
(d, J=11.35 Hz, 2 H) 3.69
(q, J=5.87 Hz, 2 H) 4.09 (s, 2 H) 4.44 (s, 3 H) 7.18 - 7.24 (m, 1 H) 7.31 (t,
J=7.63 Hz, 2 H) 7.35 - 7.42
(m, 2 H) 7.51 (br. s., 1 H) 7.93 (dd, J=8.22, 1.17 Hz, 1 H) 8.11 (d, J=1.17
Hz, 1 H) 8.43 (d, J=8.22 Hz, 1
H) 9.04 (br. s., 1 H) 12.15 (s, 1 H); HPLC 99% at 254 nm, Rt 2.063 minutes;
HRMS m/z 482.2817
(M+H)4.
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Example 36 (methyl oxadiazole from cyanide)
0-N
NC a N HO H H
Nµ 11-1I
CI N N N 411,
-N NH2OH H DIPEA H2N N 41 1) Ac2.
Microwave /
HN
-N
L\- Et0H
2) DBU
Me0H HN
N3 HN
LIN3
N3
Example 37 Example 36
[0175] Hydroxylamine hydrochloride (32.7 mg, 0.471 mmol) was added to a
solution of Example 37 (2-
benzy1-44(3-(piperidin-1-yl)propyl)amino)-9H-pyrimido[4,5-b]indole-7-
carbonitrile, 50 mg, 0.118 mmol)
in Et0H (1.5 mL) followed by DIPEA (84 pL, 0.483 mmol) to give a light yellow
suspension. After
stirring for 2.5 days at room temperature and 6 hours at 75 C, the solvent was
evaporated and water (3
mL) was added and after stirring for 30 min., the solid was collected, washed
with water (3 x 1 mL) and
the solid material was dried at 35 C under high vacuum until constant weight
to give (Z)-2-benzyl-N'-
hydroxy-44(3-(piperidin-1-yl)propyl)amino)-9H-pyrimido[4,5-b]indole-7-
carboximidamide - HCI (53 mg,
0.107 mmol, 91% yield) as a tan solid; 1H NMR (400 MHz, DMSO-d6) 6 ppm 1.58 -
1.85 (m, 6 H) 1.98 -
2.10 (m, 2 H) 2.69- 2.90 (m, 2 H) 2.94 - 3.15 (m, 2 H) 3.34 - 3.46 (m, 2 H)
3.59 - 3.74 (m,2 H) 4.05 (s, 2
H) 5.84 (br. s., 2 H) 7.15- 7.24 (m, 1 H) 7.29 (t, J=7.4 Hz, 3 H) 7.37 (d,
J=7.4 Hz, 2 H) 7.55 (dd, J=8.2,
1.2 Hz, 1 H) 7.72 (d, J=1.2 Hz, 1 H) 8.25 (d, J=8.6 Hz, 1 H) 9.47 (d, J=7.4
Hz, 1 H) 9.56 (s, 1 H) 11.88
(s, 1 H); HRMS rrilz 458.2662 (M+H)'-. HPLC 95.4% @ 220 nm and 97.4% @ 254 nm,
RT = 1.40
minutes.
[0176] Acetic anhydride (0.917 mL, 9.72 mmol) was added to (Z)-2-benzyl-N'-
hydroxy-4-((3-(piperidin-
1-yl)propyl)amino)-9H-pyrimido[4,5-b]indole-7-carboximidamide, HCI (0.040 g,
0.081 mmol) to give a
tan suspension and the mixture was heated by microwaves to 140 C for 30
minutes. The solvent was
then evaporated and the residue purified by flash chromatography to give 36 mg
(85% yield) of 1-(2-
benzy1-7-(5-methyl-1,2,4-oxadiazol-3-y1)-44(3-(piperidin-111)propyl)amino)-9H-
pyrimido[4,5-b]indol-9-
yl)ethanone as a tan solid which was dissolved immediately in methanol (2.3
mL) and treated with DBU
(0.021 mL, 0.138 mmol). The resulting yellow solution was heated to reflux for
30 minutes, then cool to
0 C while stirring for 1 hour. The solids were filtered and washed with cold
Me0H (2 x 0.5 mL), dried at
40 C under high vacuum until constant weight to afford Example 36: 2-benzy1-7-
(5-methyl-1,2,4-
oxadiazol-3-y1)-N-(3-(piperidin-1-yl)propy1)-9H-pyrimido[4,5-13]indol-4-amine
(20 mg, 51.3 % yield) as a
tan solid: 1H NMR (400 MHz, DMSO-d6) 6 ppm 1.38 (m, J=4.7 Hz, 2 H) 1.50 (quin,
J=5.5 Hz, 4 H) 1.80
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(quin, J=6.9 Hz, 2 H) 2.18 - 2.45 (m, 6 H) 2.67 (s, 3 H) 3.57 - 3.70 (m, 2 H)
4.04 (s, 2 H) 7.15 -7.22 (m,
1 H) 7.27 (m, J=7.6, 7.6 Hz, 2 H) 7.34 (t, J=5.9 Hz, 1 H) 7.36 - 7.40 (m, 2 H)
7.83 (dd, J=8.2, 1.4 Hz, 1
H) 8.01 (d, J=1.4 Hz, 1 H) 8.39 (d, J=8.2 Hz, 1 H) 12.02 (br. s., 1 H); HRMS
m/z 482.2663 (M+H)i .
HPLC 99.3%, RI = 1.79 minutes.
Example 37
is CO2Me
NCr NC
N poc,3
, N.2
Me0Na/Me0H
NH2 'N
0 HO
37A 37B
NC
N
NC
N H2N Et3N, Me0H
'N 140 C HN
CI .N10
37C Example 37
[0177] An orange mixture of 2-amino-6-cyano-1H-indole-3-carboxamide (0.172 g,
0.859 mmol), methyl
2-phenylacetate (0.303 mL, 2.148 mmol) and sodium methoxide 30%wt in Me0H
(0.403 mL, 2.148
mmol) in methanol (2.82 mL) was heated in a microwave tube at 140 C for 45
minutes. Then, a new
load of methyl 2-phenylacetate (0.151 mL, 1.074 mmol) and sodium methoxide
30%wt in Me0H (0.201
mL, 1.074 mmol) were added and the vial was placed in the microwave and heated
again at 140 C for
45 minutes. Then, after cooling to room temperature, AcOH (0.197 mL, 3.44
mmol) was added and the
resulting slurry was stirred at 20 C for 1 hour. The solids were filtered,
washed with Me0H (3 x 1 mL)
and dried at 20 C under high vacuum until constant weight to give Intermediate
37B: 2-benzy1-4-
hydroxy-9H-pyrimido[4,5-b]indole-7-carbonitrile (182 mg, 70.5% yield) as a tan
solid: 1H NMR (400
MHz, DMSO-d6) 6 ppm 4.03 (5, 2 H) 7.22 - 7.29 (m, 1 H) 7.30 - 7.36 (m, 2 H)
7.36 - 7.43 (m, 2 H) 7.58
(dd, J=8.2, 1.4 Hz, 1 H) 7.82 - 7.87 (m, 1 H) 8.05 (d, J=8.2 Hz, 1 H) 12.59
(br. s., 2 H); MS m/z 301.2
(M+H); HPLC 94.2% @ 220 nnn and 91.3% @ 254 nm; RI = 1.90 minutes.
[0178] A red mixture of 2-benzy1-4-hydroxy-9H-pyrimido[4,5-b]indole-7-
carbonitrile (Intermediate 37B,
0.180 g, 0.599 mmol) and phosphorus oxychloride (3.63 mL, 39.0 mmol) was
heated to 95 C and
stirred for 16 hours. After concentration to dryness on a rotovap, the
resulting dark red foam was
suspended in sat. NaHCO3 (10 mL) and stirred for 30 minutes. The solids were
collected and washed
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with water (3 x 1 mL), dried at 40 C under high vacuum until constant weight
to give Intermediate 37C:
2-benzy1-4-chloro-9H-pyrimido[4,5-b]indole-7-carbonitrile (190 mg, 99% yield)
as a tan solid which was
used directly in the next step: MS m/z 319.2 (M+H)i; HPLC 95.0% @ 220 nm and
92.3% @ 254 nm ,
RT = 2.28 minutes.
[0179] A mixture of 2-benzy1-4-chloro-9H-pyrimido[4,5-b]indole-7-carbonitrile
(Intermediate 37C, 0.190
g, 0.596 mmol), 3-(piperidin-1-yl)propan-1-amine (0.142 mL, 0.894 mmol) and
triethylamine (0.208 mL,
1.490 mmol) in Me0H (4.50 mL) was heated by microwaves to 140 C for 30
minutes. Then it was
concentrated to dryness to give 346 mg of an orange solid which was purified
by flash chromatography
to give 176 mg of a yellow solid which was suspended in ether (7 mL) and
stirred at 20 C for 1 hour.
The solids were filtered, washed with ether (3 x 1 mL) and dried at 30 C under
high vacuum until
constant weight to afford 2-benzy1-4-((3-(piperidin-1-yl)propyl)amino)-9H-
pyrimido[4,5-b]indole-7-
carbonitrile as Example 37(172 mg, 68.0% yield) as a light yellow solid: 1H
NMR (400 MHz, DMSO-d6)
6 ppm 1.30 - 1.43 (m, 2 H) 1.43 - 1.57 (m, 4 H) 1.80 (m, J=5.5 Hz, 2 H) 2.18 -
2.47 (m, 6 H) 3.62 (q,
J=6.4 Hz, 2 H) 4.04 (s, 2 H) 7.15 -7.22 (m, 1 H) 7.27 (m, J=7.4, 7.4 Hz, 2 H)
7.33- 7.39 (m, 2 H) 7.47
(t, J=5.7 Hz, 1 H) 7.62 (dd, J=8.2, 1.2 Hz, 1 H) 7.79 (d, J=1.2 Hz, 1 H) 8.43
(d, J=8.2 Hz, 1 H) 12.19 (s,
1 H); HRMS m/z 425.2448 (M+H)-'; HPLC >990/0, RT = 1.68 minutes.
Example 43
0
0 N
Pcl(PPI13)4
N
/ K2CO3
DME
*--"N Water
CI
[0180] In a 2-5 mL microwave vial was added methyl 2-benzy1-4-chloro-9H-
pyrimido[4,5-b]indole-7-
carboxylate (0.100 g, 0.284 mmol), (E)-1-(4-(4,4,5,5-tetramethy1-1,3,2-
dioxaborolan-2-yObut-3-en-1-
Apiperidine (0.113 g, 0.426 mmol), potassium carbonate (0.106 g, 0.768 mmol)
and Pd(Ph3P)4 (0.05 g,
0.044 mmol). The vial was purged with N2 (3 vacuum + refill cycles). DME (2.84
mL) and water (0.398
mL) were added and the vial was flushed with N2 (one vacuum + refill) and then
heated to 110 C while
stirred for 24 hours. After cooling, the mixture was concentrated to dryness
under reduced pressure
and the residue was purified by flash chromatography to give (E)-methyl 2-
benzy1-4-(4-(piperidin-1-
yl)but-1-en-1-y1)-9H-pyrimido[4,5-b]indole-7-carboxylate (55 mg, 0.121 mmol,
42.6% yield) as a light
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yellow solid: 1H NMR (400 MHz, DMSO-d6) 6 ppm 1.41 (s, 2 H) 1.50 -1.61 (m, 4
H) 2.30 - 2.47 (m, 4
H) 2.53 - 2.59 (m, 2 H) 2.59 -2.70 (m, 2 H) 3.91 (s, 3 H) 4.27 (s, 2 H) 7.16 -
7.24 (m, 1 H) 7.29 (t, J=7.6
Hz, 2 H) 7.34 - 7.43 (m, 4 H) 7.88 (dd, J=8.2, 1.4 Hz, 1 H) 8.07 (d, J=1.4 Hz,
1 H) 8.41 (d, J=8.2 Hz, 1
H) 12.48 (s, 1 H); HRMS m/z 455.2442 (M+H)+; HPLC 100% @220 nm and 99.4% @254
nm, RI =
1.84 minutes.
0
0
N
N
/ H2
Pd-C "N
'N
Me0H
THF
N
Example 43
[0181] A mixture of (E)-methyl 2-benzy1-4-(4-(piperidin-1-yl)but-1-en-1-y1)-9H-
pyrimido[4,5-b]indole-7-
carboxylate (20 mg, 0.044 mmol) and Pd-C 10%wt. (50% wet) (23.41 mg) in Me0H
(2 mL) and THF (2
mL) was treated with hydrogen for 17 hours. The reaction mixture was diluted
with DCM (3 mL),
filtered, rinsed with Me0H (2 x 2 mL) and then with DCM (2 x 2 mL) and
concentrated to dryness to
give 19 mg as a light yellow solid which was purified by flash chromatography
to give a white solid (14
mg) which was treated with CH3CN (2 mL). After stirring the white suspension
at 20 C for 1 hour, the
solid was filtered, washed with CH3CN (1 x 1 mL) and dried at 40 C under high
vacuum until constant
weight to afford the compound of Example 43 as methyl 2-benzy1-4-(4-(piperidin-
1-yl)buty1)-9H-
pyrimido[4,5-b]indole-7-carboxylate (14.4 mg, 71.7 % yield) as a white solid:
1H NMR (400 MHz,
DMSO-d6) 6 ppm 1.36 (m, J=5.5 Hz, 2 H) 1.45 (quin, J=5.5 Hz, 4 H) 1.57 (quin,
J=7.3 Hz, 2 H) 1.83 (dt,
J=14.9, 7.4 Hz, 2 H) 2.18 -2.31 (m, 6 H) 3.20 - 3.28 (m, 2 H) 3.91 (s, 3 H)
4.26 (s, 2 H) 7.16 -7.22 (m,
1 H) 7.28 (t, J=7.4 Hz, 2 H) 7.32 - 7.38 (m, 2 H) 7.90 (dd, J=8.2, 1.2 Hz, 1
H) 8.09 (d, J=1.2 Hz, 1 H)
8.25 (d, J=8.2 Hz, 1 H) 12.48 (br. s., 1 H); HRMS m/z 457.2598 (M+H)+; HPLC
>99.5%, RT = 1.75
minutes.
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Example 44
Me02C N,11
Me02C 41,
1- Et3N 3- Nal, Na2CO3 /
-14 Me HN
CI -/4 Bad CI HO
2- TFA
Intermediate 27D Example 44 HO
0
[0182] A mixture of methyl 2-benzy1-4-chloro-9H-pyrimido[4,5-b]indole-7-
carboxylate (0.100 g, 0.284
mmol), Et3N (0.079 mL, 0.569 mmol) and tert-butyl (3-
aminopropyl)(methyl)carbamate (0.080 g, 0.426
mmol) in Me0H (1 mL) was heated in a microwave oven at 140 C for 40 minutes.
The solvent was
removed under reduced pressure and the residue was purified by flash
chromatography to give
0.092mg of crude Boc derivative which was used directly in the next step. TEA
(1.0 ml, 12.98 mmol)
was added dropwise to a cold suspension of methyl 2-benzy1-44(3-((tert-
butoxycarbonyl)(methyl)amino)propyl)amino)-9H-pyrimido[4,5-b]indole-7-
carboxylate (0.092 g, 0.183
mmol) and the mixture was allowed to warm to room temperature over 30 minutes.
After dilution with
toluene, the solvent was removed under reduced pressure and then the residue
was diluted with Et0Ac
to produce 85mg of a solid used directly in the next step: HRMS m/z 404.2091
(M+H).
[0183] A mixture of methyl 2-benzy1-4-((3-(methylamino)propyl)amino)-9H-
pyrimido[4,5-b]indole-7-
carboxylate 2,2,2-trifluoroacetate (0.020 g, 0.039 mmol), sodium carbonate
(8.81 mg, 0.083 mmol),
sodium iodide (1.448 mg, 9.66 pmol) and 2-(2-(2-chloroethoxy)ethoxy)ethanol
(6.46 pl, 0.044 mmol)
was heated at 70 C in acetone (0.2 mL). After 15
hours, a second portion of 2-(2-(2-
chloroethoxy)ethoxy)ethanol reagent (6.46 pl, 0.044 mmol) was added and the
mixture was heated
again at 70 C for 15 hours. After cooling to room temperature, the mixture was
diluted with Et0Ac,
washed with water, dried over anh. MgSO4, filtered and the solvent evaporated
to give a residue which
was purified by flash chromatography to afford 9 mg of Example 44: 1H NMR (400
MHz, DMSO-d6) 6
ppm 1.74- 1.85 (m, 2 H) 2.25 (br. s., 3 H) 2.55 (br. s., 2 H) 3.32 -3.36 (m, 4
H) 3.39- 3.46 (m, 6 H)
3.51 (t, J=5.87 Hz, 2 H) 3.64 (q, J=6.52 Hz, 2 H) 3.88 (s, 3 H) 4.04 (s, 2 H)
4.53 (br. s., 1 H) 7.18 (t,
J=7.40 Hz, 1 H) 7.28 (t, J=7.63 Hz, 2 H) 7.37 (d, J=7.04 Hz, 2 H) 7.55 (t,
J=5.28 Hz, 1 H) 7.82 (dd,
J=8.22, 1.17 Hz, 1 H) 7.99 (s, 1 H) 8.27 (d, J=8.22 Hz, 1 H) 12.05 (s, 1 H);
HRMS m/z 536.2855
(M+H); HPLC RT 2.035 minutes.
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Examples 45 and 51
0
0
0 N
'0
0
N
HH/ N,S) Me0H
Microwave.. -N
CI HIV\ HN\ \
Intermediate 2713 Me .m. N84
Example 45 Example 81
[0184] In a 2-5 mL microwave vial was added methyl 2-benzy1-4-chloro-9H-
pyrimido[4,5-b]indole-7-
carboxylate (0.050 g, 0.142 mmol) and N1-(3-aminopropyI)-N1-methylpropane-1,3-
diamine (0.115 mL,
0.711 mmol) in Me0H (2.000 mL, 49.4 mmol) to give a tan suspension. The vial
was placed in the
microwave and heated to 140 C for 30 min. After 30 minutes, the mixture was
concentrated to dryness
on a rotavap. and the residue was purified by flash chromatography and
lyophilized from CH3CN to
afford two distinct products: Example 45 as the mono-N-alkylated product:
methyl 4-((3-((3-
aminopropyl)(methyl)amino)propyl)amino)-2-benzy1-9H-pyrimido[4,5-b]indole-7-
carboxylate (44 mg,
67.2% yield) as a white solid; 1H NMR (400 MHz, DMSO-d6) 6 ppm 1.44 (dt,
J=13.8, 6.6 Hz, 2 H) 1.72
(dt, J=13.7, 6.8 Hz, 2 H) 2.11 (s, 3 H) 2.24 -2.30 (m, 2 H) 2.33 (t, J=6.7 Hz,
2 H) 2.47 (br. s., 2 H) 3.51 -
3.61 (m, 2 H) 3.76 -3.85 (m, 3 H) 3.97 (s, 2 H) 7.08 -7.15 (m, 1 H) 7.17- 7.24
(m, 2 H) 7.27 -7.34 (m,
2 H) 7.50 (t, J=5.3 Hz, 1 H) 7.76 (dd, J=8.2, 1.6 Hz, 1 H) 7.92 (d, J=1.6 Hz,
1 H) 8.20 (d, J=8.2 Hz, 1 H);
MS m/z 461.2 (M+H)+; HPLC >99%, RT = 1.63 minutes.
[0185] Example 51 as a bis-alkylated product: dimethyl 4,4'-
(((methylazanediy1)bis(propane-3,1-
diy1))bis(azanediy1))bis(2-benzy1-9H-pyrimido[4,5-b]indole-7-carboxylate) (3.7
mg, 6.71% yield) as a
light yellow solid: 1H NMR (400 MHz, DMSO-d6) 6 ppm 1.18- 1.29 (m, 4 H) 1.79-
1.91 (m, 4 H) 2.25
(s, 3 H) 3.58 - 3.71 (m, 4 H) 3.84 (s, 6 H) 3.97 (s, 4 H) 7.07 - 7.16 (m, 2H)
7.22 (t, J=7.4 Hz, 4 H) 7.29 -
7.34 (m, 4 H) 7.53 (t, J=5.3 Hz, 2 H) 7.77 (dd, J=8.2, 1.4 Hz, 2 H) 7.96 (d,
J=1.4 Hz, 2 H) 8.22 (d, J=8.2
Hz, 2 H) 12.01 (s, 2H); MS nn/z 776.3 (M+H)+; HPLC 94.6% @ 220 nm and 93.8% @
254 nm, RI =
2.01 minutes.
Example 52
0 0
"=0 N N *
/ Etisl / HN-43
0 "14 HIsk
DMF HN\ \/sfNHy
Me 0
Example 45 Example 52
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[0186] 2 ,5-Dioxopyrrolidin-1-y1-5-((3aS,4S,6aR)-2-oxohexahyd ro-1 H-th
ieno[3,4-d]imidazol-4-
Apentanoate (12.01 mg, 0.035 mmol) was added to a solution of methyl 44(3-((3-
aminopropyl)(methyl)amino)propyl)amino)-2-benzy1-9H-pyrimido[4,5-b]indole-7-
carboxylate (Example
45, 15 mg, 0.033 mmol) and triethylamine (6.81 p L, 0.049 mmol) in DMF (750
pL, 9.69 mmol) to give a
light yellow solution. After stirring at 20 C for 1 hour the mixture was
concentrated to dryness and the
residue was purified by flash chromatography to give a light yellow foam. The
foam was suspended in
Et20 (1 mL) and stirred for 30 minutes and the solid was collected, washed
with Et20 (2 x 0.5 mL) and
dried at 20 C under high vacuum until constant weight to give the compound of
Example 52: methyl 2-
benzy1-4-((3-(methyl(3-(5-((3aS,4S,6a R)-2-oxohexahydro-1 H-thieno[3,4-
d]imidazol-4-
yl)pentanamido)propyl)amino)propyl)am ino)-9H-pyrimido[4 ,5-13] indole-7-
carboxylate (17 mg, 76% yield)
as a light yellow solid: 1H NMR (400 MHz, DMSO-d6) 6 ppm 1.17- 1.34 (m, 3 H)
1.36- 1.51 (m, 2 H)
1.51 -1.64 (m, 3 H) 1.78 (dt, J=13.5, 6.6 Hz, 2 H) 2.02 (t, J=7.4 Hz, 2 H)
2.17 (s, 3 H) 2.32 (t, J=7.0 Hz,
2 H) 2.40 (t, J=6.7 Hz, 2 H) 2.55 (d, J=12.3 Hz, 1 H) 2.77 (dd, J=12.3, 5.1
Hz, 1 H) 2.99 - 3.11 (m, 3 H)
3.58 - 3.68 (m, 2 H) 3.88 (s, 3 H) 4.04 (s, 2 H) 4.08 (m, J=4.9, 4.9, 2.3 Hz,
1 H) 4.26 (dd, J=7.6, 5.3 Hz,
1 H) 6.34 (s, 1 H) 6.40 (s, 1 H) 7.14 - 7.22 (m, 1 H) 7.27 (t, J=7.4 Hz, 2H)
7.37 (d, J=7.0 Hz, 2 H) 7.54
(t, J=5.5 Hz, 1 H) 7.74 (t, J=5.5 Hz, 1 H) 7.82 (dd, J=8.2, 1.6 Hz, 1 H) 7.99
(d, J=1.6 Hz, 1 H) 8.27 (d,
J=8.2 Hz, 1 H) 12.05 (s, 1 H); MS m/z 687.3 (M+H)+; HPLC >99.5%, RT = 1.70
minutes.
Example 53
[0187] Intermediate 53A was prepared from commercial ethyl 2(3-tolyl)acetate.
It was then converted
to Intermediate 53B as described for Examples 15, 45 and 47. Then, the azirine
portion was developed
according to the description provided for Example 25. The last step was based
on Example 52.
me020 0, 1- Me0Ne, Me0H Me02C N N
p...cH3 1- NBS, (Bz0)2, CCI4 0Bn, NH2 2- POCI3,90*C, 2h /
11-ir cr
Et03C 2- NMO, diox aere EtO3C 0 3- M I-1 eN((C2)3NH
n 2)2, Me0H, MW HN OB
N2N
3- e) Me3SICF3, CsF 4- (Boe)20,
Et3N, DCM-Me0H CF3
Ethyl 2-( b)TBAF, THF3-tolyl)acetate Intennediate 53A Intermediate
15B
4- FInBr, K2CO3, ACN
Intermediate: -31; \ ---NHBoe
H
21i FID2,,ApPd/E: Me 04C Ni.µ Dcms-',' is N, Nc'F N M02C
NH e p,/ N,N
Etpl CF3 HN-"
NH
3- NH2OH HCI 6-12, Et3N, DCM HN HN
Pyridine, Me0H
7- TFA, DCM Me DMF \
4- TsCl. DMAP.NJN
Et3N, DCM 0 0
Intermediate 53C \ Example 53
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[0188] A mixture of ethyl 2-(m-tolyl)acetate (4.8 g, 26.9 mmol), NBS (5.27 g,
29.6 mmol) and benzoyl
peroxide (0.110 g, 0.454 mmol) was brought to reflux in CCI4 (28 mL). After 5
hours, the reaction was
cooled to 5 C, filtered and the solvent removed. Purification by flash
chromatography using
ethylacetate-hexane gave 3.8 g of the corresponding bromobenzyl derivative: 1H
NMR (400 MHz,
DMSO-d6) 6 ppm 1.19 (t, J=6.70 Hz, 3 H) 3.67 (s, 2 H) 4.09 (q, J=6.70 Hz, 2 H)
4.69 (s, 2 H) 7.15 -
7.25 (m, 1 H) 7.27 - 7.42 (m, 3 H); this material was used directly in the
next step.
[0189] A mixture of ethyl 2-(3-
(bromomethyl)phenyl)acetate (8.11 g, 31.5 mmol) and 4-
methylmorpholine 4-oxide hydrate (5.54 g, 47.3 mmol) in 1,4-dioxane (110 mL)
was heated to 100 C
for 1.5 hour. After cooling to room temperature, the volume of solvent was
reduced to half and then
diluted with Et20:Et0Ac (1:1, 120 mL), washed with water (50 mL) and the
aqueous layer extracted
with Et0Ac (60 mL). The combined organic layers were washed with water (2 x 50
mL) and then with
brine (50 mL). The organic layer was dried over anh. MgSO4, filtered and
concentrated to give 4.72 g
of a light yellow oil which was purified by flash chromatography to afford 2-
(3-formylphenyl)acetate
(2.91 g, 48% yield) as a light yellow oil: 1H NMR (400MHz, DMSO-d6) 6 ppm 1.19
(t, J=7.0 Hz, 3 H)
3.81 (s, 2 H) 4.09 (q, J=7.0 Hz, 2 H) 7.52 - 7.65 (m, 2 H) 7.79 - 7.85 (m, 2
H) 10.00 (s, 1 H); MS m/z
207.2 (M+H)+; HPLC 99%, RI = 1.67 minutes.
[0190] Trimethyl(trifluoromethyl)silane (3.13 mL, 21.20 mmol) was added to a
mixture of ethyl 2-(3-
formylphenyl)acetate (2.91 g, 15.14 mmol) and cesium fluoride (0.161 g, 1.060
mmol) in DMF (20.19
mL) cooled to 0 - 5 C. After stirring for 1.5 hour, a solution of TBAF 1M in
THF (15.14 mL) was added.
The resulting yellow solution was stirred at 0 - 5 C and after 30 minutes, the
mixture was poured into
water (150 mL) and extracted with MTBE (1 x 150 mL, then 2 x 100 mL). The
combined organic layers
were washed with water (1 x 150 mL then 1 x 100 mL) and with brine (100 mL)
and then the organic
layer was dried over anh. MgSO4, filtered and concentrated to give 3.84 g as a
light orange oil which
was purified by flash chromatography to give ethyl 2-(3-(2,2,2-trifluoro-1-
hydroxyethyl)phenyl)acetate
(663 mg, 16% yield) as a colorless oil: 1H NMR (400 MHz, DMSO-d6) 6 ppm 1.17
(t, J=7.0 Hz, 3 H)
3.68 (s, 2 H) 4.08 (q, J=7.0 Hz, 2 H) 5.13 (q, J=7.4 Hz, 1 H) 6.82 (s, 1 H)
7.24 - 7.31 (m, 1 H) 7.37 (t,
J=7.2 Hz, 3 F); MS m/z 263.1 (M-FH)+; HPLC 93.7% @ 220 nm, RI = 1.82 minutes.
[0191] Benzyl bromide (0.330 mL, 2.78 mmol) was added to a mixture of ethyl 2-
(3-(2,2,2-trifluoro-1-
hydroxyethyl)phenyl)acetate (0.648 g, 2.471 mmol) and K2CO3 (1.059 g, 7.66
mmol) in acetonitrile
(17.00 mL) and the mixture was stirred while heated to reflux (75 - 80 C) for
24 hours. After cooling
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and concentration to dryness, the residue was purified by flash chromatography
to give Intermediate
53A: ethyl 2-(3-(1-(benzyloxy)-2,2,2-trifluoroethyl)phenyl)acetate (686 mg,
79% yield) as a colorless oil:
MS m/z 353.2 (M+H)1; HPLC 99.7%, RT = 2.19 minutes.
[0192] A mixture of Intermediate 15B (0.310 g, 1.329 mmol), ethyl 2-(3-(1-
(benzyloxy)-2,2,2-
trifluoroethyl)phenyl)acetate (0.679 g, 1.927 mmol) and Intermediate 53A and
sodium methoxide
30%wt. in Me0H (0.524 mL) in methanoL (3.23 mL) to give a thin brown
suspension which was heated
in a microwave apparatus to 140 C for 1 hour. After cooling and dilution with
Me0H (0.75 mL) and
AcOH (0.167 mL, 2.92 mmol), the resulting suspension was stirred at 20 C for 2
hours. The solid was
then collected and washed with Me0H (4 x 0.5 mL), then dried at 40 C under
high vacuum until
constant weight to afford methyl 2-(3-(1-(benzyloxy)-2,2,2-
trifluoroethyl)benzyI)-4-hydroxy-9H-
pyrimido[4,5-b]indole-7-carboxylate (399 mg, 57.6% yield) as a tan solid: 1H
NMR (400 MHz, DMSO-
d6) 6 ppm 3.87 (s, 3 H) 4.09 (5, 2 H) 4.45 - 4.57 (m, 2 H) 5.23 (q, J=7.2 Hz,
1 H) 7.20 - 7.29 (m, 5 H)
7.36- 7.51 (m, 3 H) 7.52 (s, 1 H) 7.84 (dd, J=8.2, 1.4 Hz, 1 H) 8.01 (d, J=1.4
Hz, 1 H) 8.03 (d, J=8.2
Hz, 1 H) 12.46 (br. s., 1 H) 12.56 (br. s., 1 H); MS m/z 522.2 (M+H)+; HPLC
92.7% @ 220 nm and
91.7% @ 254 nnn, RT = 2.20 minutes.
[0193] A mixture of methyl 2-(3-(1-(benzyloxy)-2,2,2-trifluoroethyl)benzyI)-4-
hydroxy-9H-pyrimido[4,5-
b]indole-7-carboxylate (0.395 g, 0.757 mmol) in phosphorus oxychloride (6 mL,
64.4 mmol) was heated
to 90 C for 2 hours then after cooling, it was concentrated to dryness to give
650 mg as a brown foam
which was suspended in sat. NaHCO3 (15 mL) and stirred for 1 hour. The solid
was filtered, washed
with water (3 x 2 mL) and dried at 40 C under high vacuum until constant
weight to afford methyl 2-(3-
(1-(benzyloxy)-2,2,2-trifluoroethyl)benzy1)-4-chloro-9H-pyrimido[4,5-b]indole-
7-carboxylate (375 mg,
92% yield) as a tan solid and used as such in the next step: MS m/z 540.2
(M+H)4; HPLC 92%, RT
2.51 minutes.
[0194] A mixture of methyl 2-(3-(1-(benzyloxy)-2,2,2-trifluoroethyl)benzyI)-4-
chloro-9H-pyrimido[4,5-
b]indole-7-carboxylate (0.375 g, 0.695 mmol) and N1-(3-aminopropyI)-N1-
methylpropane-1,3-diamine
(0.784 mL, 4.86 mmol) in Me0H (9.83 mL, 243 mmol) was heated in a microwave
oven to 140 C for 30
minutes. Then, after concentration to dryness under reduced pressure the
resulting brown oil was
purified by flash chromatography to afford methyl 4-((3-((3-
aminopropyl)(methyl)amino)propyl)amino)-2-
(3-(1-(benzyloxy)-2,2,2-trifluoroethyl)benzy1)-9H-pyrimido[4,5-b]indole-7-
carboxylate (288 mg, 63.9%
yield) as a yellow foam: 1H NMR (400 MHz, DMSO-d6) 6 ppm 1.48 (dt, J=14.0, 6.9
Hz, 2 H) 1.75 (quin,
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J=6.7 Hz, 2 H) 2.14 (s, 3 H) 2.24 -2.42 (m, 4 H) 2.52 - 2.60 (m, 2 H) 3.61 (q,
J=6.3 Hz, 2 H) 3.88 (s, 3
H) 4.10 (s, 2 H) 4,49 (s, 2 H) 5.17 (q, J=7.0 Hz, 1 H) 7.18 - 7.30 (m, 5 H)
7.31 - 7.36 (m, 1 H) 7.39 (t,
J=7.6 Hz, 1 H) 7.44 - 7.53 (m, 2 H) 7.58 (t, J=5.3 Hz, 1 H) 7.84 (dd, J=8.2,
1.4 Hz, 1 H) 8.00 (d, J=1.4
Hz, 1 H) 8.28 (d, J=8.2 Hz, 1 H); MS m/z 649.3 (M+H)+; HPLC 97.6% @ 220 nm and
95.5% @
254 nm, RT = 1.96 minutes.
[0195] A solution of di-tert-butyl dicarbonate (0.124 mL, 0.533 mmol) in DCM
(1 mL) was added slowly
to a mixture of methyl 44(3-((3-aminopropyl)(methypamino)propyl)amino)-2-(3-(1-
(benzyloxy)-2,2,2-
trifluoroethyl)benzyl)-9H-pyrimido[4,5-b]indole-7-carboxylate (0.288 g, 0.444
mmol) and triethylamine
(0.074 mL, 0.533 mmol) in DCM (3 mL) and Me0H (2 mL) to give a yellow
solution. After stirring at
20 C for 45 minutes, the solution was concentrated to dryness to give 373 mg
as a yellow foam which
was purified by flash chromatography to afford Intermediate 53B as methyl 2-(3-
(1-(benzyloxy)-2,2,2-
trifluoroethyl)benzy1)-4-((3-((3-((tert-
butoxycarbonyl)amino)propyl)(methyl)amino)propyl)amino)-9H-
pyrimido[4,5-b]indole-7-carboxylate (306 mg, 92% yield) as a yellow foam: 1H
NMR (400 MHz, DMSO-
d6) 6 ppm 1.33 (s, 9 H) 1.52 (dt, J=14.1, 7.0 Hz, 2 H) 1.67 - 1.81 (m, 2 H)
2.12 (s, 3 H) 2.28 (t, J=7.2
Hz, 2 H) 2.34 (t, J=6.8 Hz, 2 H) 2.92 (q, J=6.7 Hz, 2 H) 3.54 - 3.67 (m, 2 H)
3.88 (s, 3 H) 4.10 (s, 2 H)
4.49 (s, 2 H) 5,17 (q, J=6.8 Hz, 1 H) 6.76 (t, J=5.5 Hz, 1 H) 7.16 - 7.30 (m,
5 H) 7.31 - 7.36 (m, 1 H)
7.39 (t, J=7.6 Hz, 1 H) 7.43 - 7.51 (m, 2 H) 7.53 (t, J=5.3 Hz, 1 H) 7.83 (dd,
J=8.4, 1.4 Hz, 1 H) 8.00 (d,
J=1.4 Hz, 1 H) 8.27 (d, J=8.2 Hz, 1 H) 12.06 (s, 1 H); MS m/z 749.3 (M+H)+;
HPLC 97,7%, RI = 2.06
minutes.
[0196] A mixture of methyl 2-
(3-(1-(benzyloxy)-2,2,2-trifluoroethyl)benzyI)-4-((3-((3-((tert-
butoxycarbonyl)amino)propyl)(methyl)amino)propyl)amino)-9H-pyrimido[4,5-
b]indole-7-carboxylate
(0.306 g, 0.409 mmol) and Pd-C 10%wt (50% wet) (0.304 g, 0.143 mmol) in Me0H
(9.92 mL) was
treated with hydrogen at 20 C for 22 hours. Then, the reaction mixture was
filtered over Celite, the
cake was rinsed with Me0H (2 x 10 mL) and with DCM:Me0H (1:1, 2 x 10 mL) then
concentrated to
dryness on rotovap to give 226 mg of an oil was purified by flash
chromatography to afford methyl 4-((3-
((3-((tert-butoxycarbonyl)amino)propyl)(methyl)amino)propyl)amino)-2-(3-(2,2,2-
trifluoro-1-
hydroxyethyl)benzy1)-9H-pyrimido[4,5-13]indole-7-carboxylate (202 mg, 75%
yield) as a white foam:
1H NMR (400 MHz, DMSO-d6) 6 ppm 1.33 (s, 9 H) 1.48- 1.62 (m, 2 H) 1.71 - 1.85
(m, 2 H) 2.16 (s, 3
H) 2.25 - 2.35 (m, 2 H) 2.39 (t, J=6.8 Hz, 2 H) 2.86 - 3.00 (m, 2 H) 3.56 -
3.70 (m, 2 H) 3.88 (s, 3 H)
4.06 (s, 2 H) 5.01 - 5.14 (m, 1 H) 6.77 (m, J=6.3 Hz, 2 H) 7.30 (d, J=4.7 Hz,
2 H) 7.36 - 7.42 (m, 1 H)
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7.49 (s, 1 H) 7.51 -7.57 (m, 1 H) 7.82 (dd, J=8.2, 1.2 Hz, 1 H) 7.99 (d, J=1.2
Hz, 1 H) 8.26 (d, J=8.2
Hz, 1 H) 12.06 (br. s., 1 H); MS m/z 659.2 (M+H)+; HPLC >97%, RT = 1.90
minutes.
[0197] A mixture of methyl 4-((34(3-((tert-
butoxycarbonyl)amino)propyl)(methyl)amino)propyl)amino)-
2-(3-(2,2,2-trifluoro-1-hydroxyethyl)benzy1)-9H-pyrimido[4,5-b]indole-7-
carboxylate (0.200 g, 0.304
mmol) and Dess-Martin periodinane reagent (0.567 g, 1.336 mmol) in DCM (7.50
mL) were stirred at
20 C for one hour. After evaporation to dryness, the residue was purified by
flash chromatography to
afford methyl 4-((3-((3-((tert-
butoxycarbonyl)amino)propyl)(methyl)amino)propyl)amino)-2-(3-(2,2,2-
trifluoroacetyl)benzy1)-9H-pyrimido[4,5-b]indole-7-carboxylate (181 mg, 91%
yield) as a yellow foam: 1H
NMR in DMSO-d6 is consistent with the desired product but complicated due to
presence of hydrate
form: MS m/z 657.3 (M+H)-E; HPLC 96.0% @ 220 nm and 95.3% @ 254 nm, RT 1.87
and 1.96 (ketone
+ hydrate) minutes.
[0198] A mixture of methyl 4-((3-((3-((tert-
butoxycarbonyl)amino)propyl)(methyl)amino)propyl)amino)-
2-(3-(2,2,2-trifluoroacetyl)benzy1)-9H-pyrimido[4,5-b]indole-7-carboxylate
(0.180 g, 0.274 mmol),
hydroxylamine hydrochloride (0.023 g, 0.329 mmol) and pyridine (0.355 mL) in
Me0H (1.9 mL) was
heated in a microwave oven to 65 C for 48 hours. After concentration of the
reaction mixture to
dryness, a solution of the residue was washed with sat. NaHCO3 (15 mL), the
organic layer was dried
over anh. MgSO4, filtered and concentrated to dryness to afford methyl 44(34(3-
((terl-
butoxycarbonyl)amino)propyl)(methyl)amino)propyl)annino)-2-(3-(2,2,2-trifluoro-
1-
(hydroxyimino)ethyl)benzy1)-9H-pyrimido[4,5-b]indole-7-carboxylate (184 mg,
100% yield) as a light
yellow foam: MS m/z 672.3 (M+H)+; HPLC 96.0% @ 220 nm and 91.4% @ 254 nm, RT =
2.01 minutes.
[0199] Ts-CI (0.060 g, 0.315 mmol) was added portion-wiseto a mixture of
methyl 4-((3-((3-((tert-
butoxycarbonyl)amino)propyl)(methyl)amino)propyl)amino)-2-(3-(2,2,2-trifluoro-
1-
(hydroxyimino)ethyl)benzy1)-9H-pyrimido[4,5-b]indole-7-carboxylate (0.184 g,
0.274 mmol), DMAP (3.35
mg, 0.027 mmol) and triethylamine (0.048 mL, 0.342 mmol) in DCM (12 mL) to
give a tan solution.
After 1 hour, the reaction mixture was diluted with DCM (12 mL), washed with
water (3 x 12 mL) and
the organic layer was dried over anh. MgSO4, filtered and concentrated to
dryness to afford methyl 4-
((34(3-((tert-butoxycarbonyl)amino)propyl)(methyl)amino)propyl)amino)-2-(3-
(2,2,2-trifluoro-1-
((tosyloxy)imino)ethyl)benzyI)-9H-pyrimido[4,5-b]indole-7-carboxylate (217 mg,
96% yield) as a tan
foam: MS m/z 826.2 (M+H)+; HPLC 93.2% @ 220 nm and 91.3% @ 254 nm, RT = 2.20
minutes.
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[0200] A solution of methyl 44(34(3-((tert-
butoxycarbonyl)amino)propyl)(methypamino)propyl)amino)-
2-(3-(2,2,2-trifluoro-1-((tosyloxy)imino)ethyl)benzyl)-9H-pyrimido[4,5-
b]indole-7-carboxylate (0.217 g,
0.263 mmol) in DCM (5.07 mL) was cooled to -78 C and ammonia (1.7 mL, 79 mmol)
was condensed
into the sealed tube. The mixture was allowed to slowly warm to 20 C and
stirred for 3 hours. After
cooling again to -78 C, the sealed tube was fitted with a septa with gas
outlet and slowly warmed to
20 C to evaporate ammonia. After 3 hours, the mixture was concentrated to
dryness and then purified
by flash chromatography to afford methyl 4-((3-((3-
((tert-
butoxycarbonyl)amino)propyl)(methyl)amino)propyl)amino)-2-(3-(3-
(trifluoromethyl)diaziridin-3-
yl)benzyI)-9H-pyrimido[4,5-b]indole-7-carboxylate (139 mg, 79% yield) as a
white foam: 1H NMR (400
MHz, DMSO-d6) 6 ppm 1.34 (s, 9 H) 1.52 - 1.62 (m, 2 H) 1.72 - 1.89 (m, 2 H)
2.08 - 2.25 (m, 3 H) 2.30
- 2.44 (m, 4 H) 2.94 (q, J=6.4 Hz, 2 H) 3.64 (q, J=6.5 Hz, 2 H) 3.88 (s, 3 H)
3.93 (d, J=8.4 Hz, 1 H) 4.04
(d, J=8.4 Hz, 1 H) 4.08 (s, 2 H) 6.78 (br. s., 1 H) 7.31 - 7.41 (m, 2 H) 7.44 -
7.50 (m, 1 H) 7.54 (t, J=5.5
Hz, 1 H) 7.59 (s, 1 H) 7.83 (dd, J=8.4, 1.4 Hz, 1 H) 7.99 (d, J=1.4 Hz, 1 H)
8.27 (d, J=8.4 Hz, 1 H) 12.07
(s, 1 H); MS m/z 671.4 (M+H)+; HPLC 98.6% @ 220 nm and 96.4% @ 254 nm, RI =
1.91 minutes.
[0201] Iodine (27.8 mg, 0.110 mmol) was added to a 5 mL round-bottomed flask
protected from light
and pre-charged with methyl 44(34(3-((tert-
butoxycarbonyl)amino)propyl)(methyl)amino)propyl)amino)-
2-(3-(3-(trifluoromethyl)diaziridin-3-y1)benzyl)-9H-pyrimido[4,5-b]indole-7-
carboxylate (70 mg, 0.104
mmol) and triethylamine (43.6 pL, 0.313 mmol) in DCM (2 mL) to give a light
yellow solution. After
stirring at 20 C for 15 minutes, the solvent was evaporated and the residue
purified by flash
chromatography to give methyl 4-((3-((3-
((tert-
butoxycarbonyl)amino)propyl)(methyl)amino)propyl)amino)-2-(3-(3-
(trifluoromethyl)-3H-diazirin-3-
yObenzy1)-9H-pyrimido[4,5-b]indole-7-carboxylate (65 mg, 0.097 mmol, 93 %
yield) as a light yellow
foam: 1H NMR (400 MHz, DMSO-d6) 6 ppnn 1.33 (s, 9 H) 1.53 (dt, J=13.8, 7.0 Hz,
2 H) 1.70 - 1.82 (m,
2 H) 2.15 (br. s., 3 H) 2.24 -2.34 (m, 2 H) 2.34 -2.42 (m, 2 H) 2.87 - 2.98
(m, 2 H) 3.55 - 3.66 (m, 2 H)
3.88 (s, 3 H) 4.10 (s, 2 H) 6.76 (br. s., 1 H) 7.14 (d, J=8.2 Hz, 1 H) 7.27
(s, 1 H) 7.43 (t, J=7.8 Hz, 1 H)
7.49- 7.58 (m, 2 H) 7.83 (dd, J=8.2, 1.4 Hz, 1 H) 7.99 (d, J=1.4 Hz, 1 H) 8.27
(d, J=8.2 Hz, 1 H) 12.06
(s, 1 H); MS nn/z 669.2 (M+H)+; HPLC 97.6% @220 nm and 97.3% @254 nm, RI =
2.18 minutes.
[0202] Trifluoroacetic acid (0.400 mL, 5.19 mmol) was added to a solution of
methyl 4-((3-((3-((tert-
butoxycarbonyl)amino)propyl)(methyl)amino)propyl)amino)-2-(3-(3-
(trifluoromethyl)-3H-diazirin-3-
yl)benzyI)-9H-pyrimido[4,5-b]indole-7-carboxylate (0.064 g, 0.096 mmol) in DCM
(4 mL) to give a light
yellow solution. After stirring at 20 C for 30 minutes, the reaction mixture
was diluted with DCM
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(15 mL), washed with sat. NaHCO3 (10 mL) and the aqueous layer was back-
extracted with DCM
(10 mL). The combined organic layers were dried over anh. MgSO4, filtered and
concentrated to
dryness to afford Intermediate 53C: methyl 44(3-((3-
aminopropyl)(methyl)amino)propyl)amino)-2-(3-(3-
(trifluoromethyl)-3H-diazirin-3-y1)benzyl)-9H-pyrimido[4,5-13]indole-7-
carboxylate (45 mg, 83% yield) as
a light yellow foam: HRMS m/z 569.2601 (M+H)+; HPLC 97.1% @220 nm and 96.9%
254 nm, RT =
1.96 minutes.
[0203] 2 ,5-Dioxopyrrolidi n-1-y1-5-((3aS,4S,6aR)-2-oxohexahyd ro-1 H-th
ieno[3,4-d]imidazol-4-
yl)pentanoate (29.2 mg, 0.085 mmol) was added to a mixture of methyl 4-((3-((3-
aminopropyl)(methyl)amino)propyl)amino)-2-(3-(3-(trifluoromethyl)-3H-diazirin-
311)benzyl)-9H-
pyrimido[4,5-b]indole-7-carboxylate (45 mg, 0.079 mmol) and triethylamine
(16.55 pL, 0.119 mmol) in
DMF (750 pL) to give a yellow solution. After stirring at 20 C for 30 minutes,
the reaction mixture was
concentrated to a light orange oil under high vacuum and the residue was
purified by flash
chromatography to give 56 mg as a white solid which was lyophilized from CH3CN
to afford the
compound of Example 53: methyl 44(3-(methyl(3-(54(3aS,4S,6aR)-2-oxohexahydro-
1H-thieno[3,4-
d]imidazol-4-yl)pentanamido)propyl)amino)propyl)amino)-2-(3-(3-
(trifluoromethyl)-3H-diazirin-3-
yl)benzyI)-9H-pyrimido[4,5-b]indole-7-carboxylate (51 mg, 0.064 mmol, 81%
yield) as a white solid: 1H
NMR (400 MHz, DMSO-d6) 5 ppm 1.18 - 1.34 (m, 3 H) 1.35 - 1.50 (m, 3 H) 1.50 -
1.64 (m, 3 H) 1.70 -
1.82 (m, 2 H) 2.02 (t, J=7.4 Hz, 2 H) 2.15 (s, 3 H) 2.26 - 2.34 (m, 2 H) 2.37
(t, J=6.7 Hz, 2 H) 2.55 (d,
J=12.5 Hz, 1 H) 2.77 (dd, J=12.1, 5.1 Hz, 1 H) 2.99 - 3.10 (m, 3 H) 3.56 -
3.66 (m, 2 H) 3.88 (s, 3 H)
4.03 -4.14 (m, 1 H) 4.10 (s, 2 H) 4.26 (dd, J=7.6, 5.3 Hz, 1 H) 6.34 (s, 1 H)
6.39 (s, 1 H) 7.14 (d, J=7.4
Hz, 1 H) 7.28 (s, 1 H) 7.44 (t, J=7.8 Hz, 1 H) 7.52 (d, J=7.8 Hz, 1 H) 7.56
(t, J=5.3 Hz, 1 H) 7.73 (t,
J=5.5 Hz, 1 H) 7.83 (dd, J=8.2, 1.4 Hz, 1 H) 8.00 (d, J=1.4 Hz, 1 H) 8.28 (d,
J=8.2 Hz, 1 H) 12.07 (s, 1
H); HRMS nn/z 795.3368 (M+H)4; HPLC 95.4% @ 220 nm and 96.2% @ 254 nm, RT =
2.06 minutes.
Example 55
Me02C
N 41104 MeNH2 MeHN
N 1110
,
-N -N
HN TFA Me0H HN
\-N/ 110 C, 66 h
Example 55
Example 27
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[0204] A solution of methyl 2-benzy1-4-((3-(piperidin-1-yl)propyl)amino)-9H-
pyrimido[4,5-b]indole-7
carboxylate (Example 27, 0.030 g, 0.066 mmol) in methylamine 2M in Me0H (10.00
mL) was placed in
a sealed the tube and heated to 110 C for 66 hours, then the mixture was
cooled to 20 C,
concentrated to dryness and purified by flash chromatography to afford 28 mg
of a colorless oil which
was suspended in ether (2 mL). After stirring the resulting suspension for 2
hours, the solids were
collected on a Buchner, the cake was washed with ether (2 x 0.5 mL) and the
product was dried at
40 C under high vacuum until constant weight to afford Example 55: 2-benzyl-N-
methy1-4-((3-(piperidin-
1-yl)propyl)amino)-9H-pyrimido[4,5-b]indole-7-carboxamide (23 mg, 77 % yield)
as a white solid: 1H
NMR (400 MHz, DMSO-d6) 6 ppm 1.38 (m, J=5.1 Hz, 2 H) 1.50 (quin, J=5.5 Hz, 4
H) 1.80 (quin, J=7.0
Hz, 2 H) 2.22 -2.41 (m, 6 H) 2.81 (d, J=4.3 Hz, 3 H) 3.58 - 3.68 (m, 2 H) 4.03
(s, 2 H) 7.15 -7.21 (m, 1
H) 7.27 (m, J=7.4, 7.4 Hz, 3 H) 7.34 - 7.40 (m, 2 H) 7.70 (dd, J=8.2, 1.4 Hz,
1 H) 7.90 (d, J=1.4 Hz, 1H)
8.27 (d, J=8.2 Hz, 1 H) 8.46 (q, J=4.3 Hz, 1 H) 11.96 (s, 1 H); HRMS rn/z
457.2708 (M+H)+; HPLC
>99.5% @ 220 nm and 98.9% @ 254 nm, RT = 1.53 minutes.
[0205] Reported HPLC retention time are for reverse-phase HPLC (Agilent, 1200
series) using the
following conditions Solvent A: MeOH:H20:TFA (5:95:0.05); Solvent B:
MeOH:H20:TFA (95:5:0.05);
flow: 3.0 mL/min.; gradient 0 to 100% B in 2.0 min; column: ZorbaxC18, 3.5
microns, 4.6 x 30 mm;
wavelength 220 nm.
Table 1. Structure, analytical HPLC retention time, LCMS data and biological
data of Examples.
Compound Structure HPLC R1 MS rn/z Biological
number (min)analytical (M+H) data (EC50)*
0
Me0
/
-N
HN
1 1.38 368.2
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Compound Structure HPLC RT MS m/z Biological
number (min)analytical (M+H)* data (EC50)*
Me0
/ N\>
¨N
2 HN 1.35 354.2
0
Me0
/
3 HN 1.55 342.2
0
Me0
/
4 1.30 314.2
HN
/
0
Me0
/ N\>
¨N
HN 1.29 328.2
¨N
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Compound Structure HPLC RT MS m/z Biological
number (min)analytical (M+H)* data (EC50)*
0
Me0
/
¨N
6 HN 1.41 354.2
0
Me0
¨N
7 HN 1.43 382.2
0
Me0
/
8 1.34 300.2
HN
H2N
0
Me0
/
¨N
9 HN 1.35 384.2
HO-y
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Compound Structure HPLC RT MS m/z Biological
number (min)analytical (M+H)* data (EC50)*
0
Me0
/
10 1.34 326.2
HN
0
Me0
/ rµ
¨N
11 HN 1.40 354.2
\-6
0
Me0
/
¨11
12 HN 1.29 370.2 A
of
0
Me0
/
13 1.45 394.2
cc
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Compound Structure HPLC RT MS m/z Biological
number (min)analytical (M+H)* data (EC50)6
NN
H
N N
/
¨N
14 HN 1.44 392.2 D
CN)
H
Me02C
¨N
15 HN
\ 1.43 459.2 E
\
Me02C Ll (-1
/--
-N
16 HN 1.78 459.2518 C
Me02C
H N
N _>_51$
/ N\i ¨N
¨N
17 HN 1.54 460.2 B
H ,
Me02C N
¨N
18 HN 2.068 464.2145 E
\
)\
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Compound Structure HPLC RT MS m/z Biological
number (min)analytical (M+H)* data (EC50)*
I
H
Me02C N
IF Br
N
/ \
---N
19 HN\_µ TEA 2.349 662.063 E
\¨N
H
Me02C N
¨N
20 HN
2.206 508.2707 D
TEA
H
Me02C N
N 411
/ \
¨N OMe
21 HN 1.78 488.2665 E
\
H
Me02C N
N Al
¨N OH
22 HN 1.68 474.2511 F
\
\¨N/ )
\
H
Me02C N
/ N .
i \
¨N 0
23 HN 2.129 472.2342 F
\
H
Me02C N
N ill
¨N me
24 HN 2.083 472.2724 E
\¨\¨ND
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Compound Structure HPLC RT MS m/z Biological
number (min)analytical (M+H)* data (EC50)6
H
N
me02C N it N=N
, \ CF3
¨NJ
HN
25 \¨\¨ND 2.05 566.2497 E
H
EtO2C N
¨N
26 HN TFA 2.152 472.2733 F
\
\¨f)
H
Me02C N
¨N
27 HN TFA 2.052 458.2598 E
H
Me02C N
N 41 Br
/ \
¨N
HN TFA
28 2.194 538.1670 E
H
Me02C N
N 411.
Me
¨N
29 HN TFA 2.142 472.2756 E
)\
Me
H
Me02C N
N it
¨N
30 HN\ TFA 2.142 472.2740 E
\¨ND
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Compound Structure HPLC RT MS m/z Biological
number (min)analytical (*M)' data (ECM)*
H
Me02C N
/ N 411 F
. \
¨N TEA
HN
31 \ 2.112 476.2499 E
\¨ND
H
Me02C N
ome
, N
, \
HN TEA
32 \¨\¨ND 2.070 488.2690 E
H 0
MeO2C N
/ N\ CF3
¨N
HN 1.761.87
(hydrates) 554.2384 E
H
Me02C N 4I OH
N
/ \ CF3
¨N
HN
2.142 554.2 E
NN
N N
N 41
/ \
35 ¨N 2.063 482.28 E
HN TFA
\
\
N-0
N N
i N Al
36 ¨N 1.79 482.2663 F
HN
\ HCI
\--Nr)
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Compound Structure HPLC RT MS m/z Biological
number (min)analytical (M+H)* data (EC50)6
NC
N
37 HN 1.68 425.2448
\¨N/
Me02C
N
¨N
38 HN 1.44 340.2
NH2
Me02C
N
39 HN 1.38 340.2
NH2
,
N *
40 HN 1.72 454.2
¨N
-iqH2
N
41 HN 1.71 482.2785
s .HCI
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Compound Structure HPLC RT MS m/z Biological
number (min)analytical (*M)' data (EC50)*
Me02C
= N
,
'N
42 1.92 459.2392
\¨f)
Me02C
N 411
'N
43 1.75 457.2598
Me02C
N *
HN HO,
44 \¨\¨Nie 0 0J 2.035 536.2867
v v
Me02C
N *
/
--N
HN
N,Me 1.63 461.2
Me02C
= N
,
"N
46 HN 1.70 474.2476
Me02C
N
'N
47 HN 1.85 512.2632
/¨\-07\¨N/¨)
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Compound Structure HPLC RT MS m/z Biological
number (min)analytical (*M)' data (EC50)*
Me02C =
N
48 Me¨N 1.74 472.2717
Me02C
N 11.
"N
49 TFA 2.161 586.2839 A
Me \
\¨N/
Me02C
N
'N
HN
50 \¨U\/ 1.65 499.2823
\¨N¨N
1-1
r(4:02C
j N =
\
51 'N 2.01 776.3
HN
\414Me
2
Me02C 11/
H --1µ1 frie Ot_
52
FINA 1.70 687.3
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Compound Structure HPLC RT MS m/z Biological
number (min)analytical (M+H)* data (EC50)*
"1/2c 1"1 * N'N HN--1(3NH
0
53 j¨NH
2.06 795.3368
Me02C
¨1%)--Me
N
54 HN 1.46 382.2 A
\¨N/
0
/
MeHN
55 HN 1.53 457.2708 A
\¨N/
[0206] The EC50 is defined as the concentration that results in a 50% increase
in CD34+CD45RA- cell
count compared to vehicle cultures (DMSO). * EC50 : A >1000 nM; B = 500-1000
nM; C = 250-500 nM;
D = 100-250; E = <100 nM; F = compound showed > 1.3 fold expansion.
Ex vivo Functional Assay:
[0207] The ex vivo functionality of expanded cells was tested using the
conventional colony-forming
units in culture (CFU-C) assay. Untreated cells or cells incubated with DMSO,
positive control or a
compound of the invention were plated in methylcellulose medium in
conventional conditions. As an
example, Compound 1 (Table 1, Example 1) expands the number of multipotent
hematopoietic
progenitors. Methylcellulose culture of 1000 CD34+ mPB cells treated with
Compound 1 for 10 days
resulted in a 5-fold increase in multilineage granulocyte erythrocyte,
macrophage and megakaryocyte
(GEMM colonies) over input cells and a 10-fold increase compared to control
cells. This suggests that
Compound 1 promotes expansion of multipotent progenitor cells.
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In vivo Functional Assay:
[0208] CD34+ mPB cells cultured with compounds of the invention engraft
immunodeficient strain
NOD scid gamma (NSG) mice. The outcome of 2,000,000 and 500,000 CD34+ mPB
cells cultured for
days with Compound 1 (Table 1, Example 1) or vehicle control conditions were
transplanted in NSG
mice. After 8 weeks post transplantation, human hematopoietic cell
reconstitution was checked in the
NSG bone marrow using antibody against human CD45. Cells treated with Compound
1 but not with
vehicle were able to engraft NSG mice. Moreover, the reconstitution of the
human myeloid and
lymphoid compartments was also confirmed, as the bone marrow cells were
positive for human CD33+
and CD19+ respectively. These results show that CD34+ mPB expanded with
Compound 1 not only
contribute to the engraftment but also retain in vivo multilineage
repopulation potential.
Combination of compounds:
[0209] CD34+ mPB cells cultured with compounds of the invention engraft
immunodeficient strain
NOD scid gamma (NSG) mice. The outcome of 2,000,000 and 500,000 CD34+ mPB
cells cultured for
10 days with Compound 1 (Table 1, Example 1) or vehicle control conditions
were transplanted in NSG
mice. After 8 weeks post transplantation, human hematopoietic cell
reconstitution was checked in the
NSG bone marrow using antibody against human CD45. Cells treated with Compound
1 but not with
vehicle were able to engraft NSG mice. Moreover, the reconstitution of the
human myeloid and
lymphoid compartments was also confirmed, as the bone marrow cells were
positive for human CD33+
and CD19+ respectively. These results show that CD34+ mPB expanded with
Compound 1 not only
contribute to the engraftment but also retain in vivo multilineage
repopulation potential.
[0210] It is understood that the examples and embodiments described herein are
for illustrative
purposes only and that various modifications or changes in light thereof will
be suggested to persons
skilled in the art and are to be included within the present discovery and
scope of the appended claims.
